qPCR 2011 Event Proceedings 5th International qPCR ... · 16:00 Single Molecule RNA FISH: novel, simple, and accurate quantitative applications for gene expression analysis Arjun

qPCR 2011.net 28th March - 1st April 2011

Symposium & Exhibition & Workshops

5th international qPCR Event, TUM, Freising-Weihenstephan, Germany

Molecular Diagnostics:from single-cells to Next Generation Sequencing

qPCR 2011 Event Proceedings(online version)

5th International qPCR SymposiumIndustrial Exhibition & Application Workshops

Molecular Diagnostics:from single-cells to Next Generation Sequencing

Editor: Michael W. Pfaffl

Physiology, Freising – WeihenstephanWeihenstephaner Berg 3

Technical University Munich (TUM)85354 Freising

Germany

www.qPCR2011.net

ISBN 9783000338403

qPCR 2011 – Online Proceedings – page 2

qPCR 2011 Sponsors

Lead Sponsors:

Gold Sponsors:

Silver Sponsors:

Further Companies in the Industrial Exhibition:

Media partners:


Table of content

qPCR 2011 Sponsors................................................................................................................................................................... 2Table of content ........................................................................................................................................................................... 3Invitation ....................................................................................................................................................................................... 4

qPCR 2011 Event Agenda............................................................................................................................................................ 5

Sunday 27th March 2011........................................................................................................................................................ 6

Agenda - Monday 28th March 2011 ........................................................................................................................................... 6Welcome & Opening of the Symposium.............................................................................................................................. 6

MIQE and QC strategies in qPCR ........................................................................................................................................ 6single-cell qPCR ................................................................................................................................................................... 6

Agenda - Tuesday 29th March 2011........................................................................................................................................... 7High throughput & digital PCR.............................................................................................................................................. 7NGS: Next Generation Sequencing...................................................................................................................................... 7RNAi - microRNA - siRNA Applications ................................................................................................................................ 8qbasePLUS: data analysis lunch seminar ............................................................................................................................ 8New qPCR Applications and Method Optimisation............................................................................................................... 8

Agenda - Wednesday 30th

March 2011 ....................................................................................................................................... 9Pre-PCR: Pre-analytical Steps ............................................................................................................................................. 9New qPCR Applications in Molecular Diagnostics................................................................................................................ 9Data Analysis: qPCR BioStatistics & BioInformatics........................................................................................................... 10GENEX: data analysis lunch seminar ................................................................................................................................. 10MIQE: state of the art & open discussion........................................................................................................................... 10RDML: state of the art & open discussion.......................................................................................................................... 10

Closing of the Symposium ................................................................................................................................................... 10

Agenda - Thursday 31st

March & Friday 1st

April 2011.......................................................................................................... 11qPCR Application Workshops.............................................................................................................................................. 11

Basic Module qPCR Application Workshop (2-days)…………………............................................................................... 11qPCR data analysis - Biostatistics & Expression Profiling (2-days)……………………....................................................... 11MIQE guidelines (1 day) & Practical primer design (1-day) ……………………............................................................... 12qPCR Workshop Sponsors:................................................................................................................................................ 12

Industrial Exhibition................................................................................................................................................................... 13

Abstracts - Oral presentations.................................................................................................................................................. 14Monday 9

thMarch 2009 ........................................................................................................................................................ 14

MIQE and QC strategies in qPCR ...................................................................................................................................... 14single-cell qPCR ................................................................................................................................................................. 15

Tuesday 29th March 2011 .................................................................................................................................................... 17High throughput & digital PCR............................................................................................................................................ 17NGS: Next Generation Sequencing.................................................................................................................................... 18RNAi - microRNA - siRNA Applications .............................................................................................................................. 20qbasePLUS: data analysis lunch seminar .......................................................................................................................... 21New qPCR Applications and Method Optimisation............................................................................................................. 21

Wednesday 30th

March 2011................................................................................................................................................. 23Pre-PCR: Pre-analytical Steps ........................................................................................................................................... 23New qPCR Applications in Molecular Diagnostics.............................................................................................................. 25Data Analysis: qPCR BioStatistics & BioInformatics........................................................................................................... 27GENEX: data analysis lunch seminar ................................................................................................................................. 29MIQE: state of the art & open discussion........................................................................................................................... 29RDML: state of the art & open discussion.......................................................................................................................... 29

Abstracts - Poster presentations.............................................................................................................................................. 30New qPCR Applications: Molecular Diagnostics & Expression Profiling P001 – P046 ..................................................... 30New qPCR Applications: Method Optimisation & Standardisation P047 – P055 .............................................................. 42qPCR Biostatistics & Bioinformatics P056 – P061............................................................................................................ 45High Throughput qPCR & digital PCR Next Genartion Sequencing P062 – P069............................................................ 46Single-cell qPCR qPCR from limited material P070 – P075 ............................................................................................. 48HRM & Methylation Studies P076 – P084 ........................................................................................................................ 50RNAi: microRNA – siRNA Applications P085 – P090 ...................................................................................................... 53

List of participants ..................................................................................................................................................................... 55


Invitation

Dear colleagues,

Dear researchers,

Dear company representatives,

On behalf of the organisation committee and the scientific board of the conference it is a great pleasure to invite you to the

qPCR 2011 Event, the 5th

International qPCR Symposium, including an Industrial Exhibition and three qPCR Application

Workshops. The symposium focus is on Molecular Diagnostics: from single-cells to Next Generation Sequencing. 71

lectures and 91 posters will be presented by international recognised experts in their application fields. The emphasis will be on

unbiased, didactic information exchange. One third of the talks will be held by invited speakers, one third of the speakers will be

selected from the submitted abstracts and one third of the oral contributions will be done by qPCR company representatives.

The poster sessions will be shown on Monday evening and around lunch time on Tuesday and Wednesday in the poster

exhibition hall below the foyer.

The qPCR Event is structured in three parts:

1. qPCR Symposium taking place March 28-30, including various talk and poster sessions

2. A parallel qPCR Industrial Exhibition with 35 company booth taking place March 28-30

3. Followed by three qPCR Workshops taking place March 31 – April 1st

powered by BioEPS GmbH and the TATAA Biocenter Sweden

The scientific organization is managed by international well-known scientists in the field of real-time PCR:

Stephen Bustin Prof. of Molecular Science, School of Medicine, London, UK

Mikael Kubista Prof. of Biotechnology, TATAA Biocenter, Sweden

Jo Vandesompele Prof. at the Center of Medical Genetics, University of Ghent, Belgium

Heinrich H. D. Meyer Prof. of Physiology, Technical University of Munich, Weihenstephan, Germany

Michael W. Pfaffl Prof. of Molecular Physiology, TUM Weihenstephan, Germany (scientific coordination)

Event organization will be supervised by Dr. Martina Reiter, BioEPS GmbH [email protected]

The qPCR 2011 event location is the central lecture hall complex and the foyer at TUM (Technical University of Munich) in

Freising Weihenstephan, Germany. The TUM and the Biotech region around Munich are part of the largest Biotech cluster in

Europe, located close to the Munich airport (MUC) directly in the heart of Bavaria.

Please enjoy the conference, the exhibition, the workshops and the social program during the following week.

Best regards

scientific coordinator


qPCR 2011 Event Agenda Overview

online agenda (HTML version) => http://agenda.qPCR2011.net online agenda (PDF version) => http://agendaPDF.qPCR2011.net

Lecture hall 14 (HS 14) Lecture hall 15 (HS 15)Foyer &

Seminar rooms S1 & S2

12:00 – 18:00Industrial Exhibition Built upSunday

27th Mach 15:00 – 18:00Arrival & Registration

10:00 – 10:30

Welcome & OpeningqPCR 2011 Symposium

Welcome by Michael W. Pfaffl &Alfons Gierl Dean WZW TUM

8:00 – 10:00Arrival & Registration

8:00 – 10:00Poster Setup

in Foyer lower level

10:30 – 13:00MIQE & QC strategies in qPCR

13:00 – 14:00

Lunch

14:00 – 18:00Single-cell qPCR

10:00 – 21:00Industrial Exhibition

Monday

28th March

18:00 – 21:00 Reception in the Industrial Exhibition18:00 – 21:00

Evening Poster Sessionin Foyer lower level

8:30 – 12:00High Throughput qPCR

& digital PCR

8:30 – 12:00RNAi:

microRNA – siRNA Applications


12:00 – 14:00

Lunch13:00 – 14:00qbasePLUS

data analysis lunch seminar

12:00 – 14:00Lunch Poster Session

Tuesday

29th March

14:00 – 18:00Next Generation Sequencing

14:00 – 18:00New qPCR Applications& Method Optimisation


19:00 – 24:00 Symposium Gala DinnerLocation: Lindenkeller, Pasta & More, Freising

International Buffet, Asian Buffet, Music & Dancing

9:00 – 12:00Pre-analytical Steps

9:00 – 12:00Biostatistics & Bioinformatics


12:00 – 14:00

Lunch

13:00 – 14:00GenEx

data analysis lunch seminar

12:00 – 14:00Lunch Poster Session

14:00 – 15:00MIQE

state of the art & open discussion13:45 – 16:30

New qPCR Applicationsin Molecular Diagnostics 15:30 – 16:30

RDMLstate of the art & open discussion

14:00 – 16:30Poster Take Downin Foyer lower level

Wednesday

30th March

16:30 – 17:00

Closing of the SymposiumHeinrich HD. Meyer & Michael W. Pfaffl

Thursday31st March

Friday1st April

9:00 - 17:00qPCR Workshops:- Basis Module qPCR Application Workshop (2-days)- qPCR data analysis: Biostatistics & Expression Profiling (2-days)- MIQE guidelines (1 day) & Practical primer design (1-day)


Agenda qPCR 2011 Event

Sunday 27th March 2011

12:00 – 18:00 Built-up for Industrial Exhibition

15:00 – 18:00 Arrival & Registration

Monday 28th March 2011

Welcome & Opening of the SymposiumLecture hall HS 14

08:00 – 10:00 Built-up for Industrial ExhibitionArrival & Registration

09:00 – 10:00 Welcome Coffee & Tea

10:00 Welcome & Opening of the SymposiumMichael W. PfafflScientific coordination of the qPCR 2009 Symposium

10:15 Welcome at the Center of Food & Life Science inFreising WeihenstephanAlfons GierlDean WZW Technical University of Munich

MIQE and QC strategies in qPCR

Time: Monday, 28/03/2011: 10:30am - 1:00pmLocation: Lecture hall 14Session Chair: Stephen Andrew Bustin, Michael W Pfaffl

10:30 MIQE: compliance and amendments

Stephen Andrew Bustin1, Jo Vandesompele2

1Barts and the London School of Medicine, QMUL, UnitedKingdom; 2Center for Medical Genetics Ghent (CMGG)Ghent University Hospital

11:00 Convenient and reliable gene expression profilingusing RealTime ready Focus Panels in combinationwith sound data-analysis using qbasePLUS

Ali Rihani1, Tom Van Maerken1, Barbara D'haene2, NurtenYigit1, Jo Vandesompele1,2, Rita Rein3; 1Center for MedicalGenetics, Ghent University, Ghent, Belgium; 2Biogazelle,Ghent, Belgium; 3Roche Diagnostics, Germany

11:30 "Stay in touch while on the bench" MIQE qPCR

Afif Abdel Nour1, Michael W. Pfaffl2

1LaSalle Beauvais, France; 2Physiology Weihenstephan,TUM, Germany

12:00 qPCR Gene Expression Assays and an ExogenousRNA control for indication of reaction inhibition in theRT-qPCR workflow

Jaakko Kurkela

Thermo Fisher, Finland

12:30 Quality control and management approaches forMolecular Diagnostics techniques used duringdiagnosis and monitoring of hematologicmalignancies

Barbara Zehentner

HematoLogics Inc, United States of America

12:45 Substantial performance discrepancies betweencommercially available kits for rt-qPCR - A systematiccomparative investigator-driven approach

Matthias W Sieber1,2, Peter Recknagel2, Florian Glaser2,Otto W. Witte1, Michael Bauer2, Ralf A. Claus2, ChristianeFrahm1

1Hans Berger Clinic for Neurology, Jena UniversityHospital, Germany; 2Anaesthesiology and Intensive CareMedicine, Jena University Hospital, Germany

13:00 – 14:00 Lunch in the student cafeteria

single-cell qPCR

Time: Monday, 28/03/2011: 2:00pm - 6:00pmLocation: Lecture hall 14Session Chair: Mikael Kubista, Anders Ståhlberg

14:00 25 years of PCR - from idea to subcellular expressionprofilingMikael KubistaTATAA BIOCENTER, Sweden

14:30 Uncovering the Diversity of Individual Cells: GeneExpression Profiling with the BioMark SystemKen LivakFluidigm Corporation, United States of America

15:00 Identification of unknown cell populations andcorrelations using single-cell gene expressionprofilingAnders StåhlbergUniversity of Gothenburg, Sweden

15:30 – 16:00 Coffee break & Networking

16:00 Single Molecule RNA FISH: novel, simple, andaccurate quantitative applications for gene expressionanalysisArjun RajUniversity of Pennsylvania, United States of America

16:30 Relationship between cell attachment and bcr-abl cellexpression heterogeneity within CML cell lines.Philip Day, Ehsan KarimianiUniversity of Manchester, United Kingdom

17:00 Digital Competitive Allele-Specific TaqMan-BasedReverse Transcription-qPCR (castRT-qPCR) for DirectDetection and Enumeration of Circulating Tumor CellsDavid Xingfei Deng, Yun Bao, Scott Sproul, Yu Wang,Fawn Wang, David Merrill, Pius Brzoska, Caifu ChenLife Technologies, United States of America

17:20 Real time PCR on slide: a new tool for quantificationof minute amounts of DNAJean-Christophe Avarre1, Angelique le Bras2, RégisMelizzi2, Maxime Rattier2, Gordana Cerovic2, ClaudeWeisbuch2, Marianna Alunni3, Martin Kantlehner3, PetraHartmann3, Reinhard Bittner3, Wolfgang Mann3

1Institut de Recherche pour le Développement,Montpellier, France; 2Genewave SAS, Paris, France;3Beckman Coulter Biomedical GmbH, Munich, Germany

17:40 Towards a Comprehensive Single Cell ExpressionProfilingHerbert AuerIRB Barcelona, Spain

18:00 – 21:00 Evening Poster Session


Tuesday 29th March 2011

High throughput & digital PCR

Time: Tuesday, 29/03/2011: 8:30am - 12:00pmLocation: Lecture hall 14Session Chair: Frank McCaughan

Kevin L Knudtson

8:30 Using Nanoscale PCR and NGS Technologies toDetect Rare Sequence Variants in a Core SettingKevin L KnudtsonUniversity of Iowa, United States of America

9:00 Customized, function tested RealTime ready assaysfor gene expression analysis in biomarker researchand early drug developmentSabine Lohmann1, Andrea Herold1, Bergauer Tobias3,Belousov Anton2, Betzl Gisela1, Dietrich Manuel1, HesselHarald5, Poignee-Heger Manuela1, Geho David4, WeisserMartin2

1Roche Applied Science, Penzberg, Germany; 2RochePharma, Penzberg, Germany; 3Roche Pharma, Basel,Switzerland; 4Roche Pharma, Nutley, USA; 5Ludwig-Maximillian-University, Institue of Pathology, Munich,Germany

9:30 Digital PCR - getting the most out of difficult clinicalsamplesFrank McCaughanMedical Research Council, United Kingdom


10:30 Digital PCR or Finding The Needle In A HaystackElena GrigorenkoLife Technologies, Inc, United States of America

11:00 Digital Analysis- a bridge to the ClinicJim WhiteNanoString Technologies, United Kingdom

11:30 High-throughput real-time PCR approach using theLightCycler®1536 instrument for genotyping and highdiscrimination of E. coli pathogroups responsible forgastrointestinal diseasesSabine Delannoy1, Lothar Beutin2, Cédric Woudstra1,Patrick Fach1

1French Agency for Food, Environmental andOccupational Health & Safety (ANSES), France; 2NRL forE. coli, Federal Institute for Risk Assessment, Germany


12:00 – 14:00 Lunch Poster Session

NGS: Next Generation Sequencing

Time: Tuesday, 29/03/2011: 2:00pm - 6:00pmLocation: Lecture hall 14Session Chair: Yuk Ming Dennis Lo, Yu-Hui Rogers

14:00 Noninvasive prenatal diagnosis using fetal DNA inmaternal plasma: from digital PCR to next-generationsequencingYuk Ming Dennis LoThe Chinese University of Hong Kong, China

14:30 Qualitative and Quantitative Analysis of RNA bySOLiD Next Generation SequencingThomas RygusLife Technologies GmbH, Germany;

15:00 Analysis of circulating nucleic acids (CNA) using NGStechnologiesBertram Brenig1, Julia Beck2, Ekkehard Schütz2, Howard BUrnovitz2

1University of Goettingen, Germany; 2Chronix BiomedicalGmbH, Germany

15:30 Improved library quantification for High ThroughputSequencing at the Wellcome Trust of Human GeneticsOxfordPaolo PiazzaWellcome Trust Centre for Human Genetics, UnitedKingdom


16:30 Application of the Next Generation SequencingTechnologies at JCVIYu-Hui RogersJ. Craig Venter Institute, United States of America

17:00 The use of qRT-PCR and high-throughputtranscriptomics for biomarker developmentMichael W Pfaffl, Irmgard Riedmaier, Heinrich HD MeyerPhysiology Weihenstephan, TUM, 85354 Freising,Germany

17:30 Deep sequencing as diagnostic tool for highlypathogenic virusesAleksandar Radonic, Andreas Kurth, Wojtek Dabbrowski,Andreas NitscheRobert Koch Institute, Germany

18:00 High Resolution Transcription Profiling using Next-Generation-Sequencing identifies Diagnostic Markersfor MalignancyRalf Horres1,3, Björn Rotter1, Peter Winter1, MichalskiChristoph2, Friess Helmut2, Jüngling Ruth1,3, Günter Kahl3,Nicolas Krezdorn1, Klaus Hoffmeier1, Jörg Kleeff2

1GenXPro GmbH, Germany; 2Chirurgische Klinik undPoliklinik, Klinikum rechts der Isar, TU München; 3GoetheUniversität Frankfurt, Institut für MolekulareBiowissenschaften

19:00 – 24:00 Symposium Gala Dinner

Location: Lindenkeller Pasta & More, Freising

International Buffet and Asian Buffet

Music and Dancing


RNAi - microRNA - siRNA Applications

Time: Tuesday, 29/03/2011: 8:30am - 12:00pmLocation: Lecture hall 15Session Chair: Jo Vandesompele, Jan Hellemans

8:30 Development of an ultra-high throughput long non-coding RNA qPCR screening systemJan Hellemans1, Pieter Mestdagh2, Steve Lefever2,Barbara D'haene1, Filip Pattyn2, Frank Speleman2, JoVandesompele1

1Biogazelle, Ghent, Belgium; 2Ghent University, Belgium

9:00 Towards blood-based cancer screening using LNA™-enhanced microRNA qPCRDitte Andreasen, Niels Tolstrup, Jacob Ulrik Fogh, SørenJensby Nielsen, Kim Bundvig Barken, Adam Baker, PeterMouritzenExiqon, Denmark

9:30 MicroRNA Target Validation with MISSION® 3'UTR LentiGoClone™ and Human MicroRNA MimicsNikos HontzeasSigma Aldrich, United States of America


10:30 Impact of different normalization strategies on miRNAprofiling experimentsSwanhild Meyer1, Sebastian Kaiser2, Carola Wagner3,Christian Thirion4, Michael Pfaffl11Physiology Weihenstephan, Technische UniversitätMünchen, Germany; 2Department of Statistics, Ludwig-Maximilians-Universität München, Germany; 3IMGMLaboratories GmbH, Martinsried, Germany; 4Friedrich-Baur-Institute and Department of Neurology, Ludwig-Maximilians-Universität München, Germany

11:00 RNAi and gene expression profiling as a tool in cancerresearchMichelle Plusquin1, An-Sofie Stevens1, Katrien DeMulder2, Frank Vanbelleghem1, Peter Ladurner2, AnnCuypers1, Tom Artois1, Karen Smeets1

1Hasselt University, Belgium; 2University of Innsbruck

11:30 Absolute and relative quantification of placentalspecific microRNAs in maternal circulation inplacental insufficiency related complicationsIlona Hromadnikova1, Katerina Kotlabova1, JindrichDoucha2, Klara Dlouha3

1Third Faculty of Medicine, Charles University Prague,Czech Republic; 2Clinic of Obstetrics and Gynecology,University Hospital Motol, Prague, Czech Republic;3Institute for the Care of the Mother and Child, Prague



qbasePLUS: data analysis lunch seminar

Time: Tuesday, 29/03/2011: 1:00pm - 2:00pmLocation: Lecture hall 15Session Chair: Barbara D'haene, Jan Hellemans

13:00 qbasePLUS 2.0: the next generation of real-time PCRdata-analysis softwareBarbara D'haene, Jo Vandesompele, Jan HellemansBiogazelle, Belgium

New qPCR Applications and Method Optimisation

Time: Tuesday, 29/03/2011: 2:00pm - 6:00pmLocation: Lecture hall 15Session Chair: Heinrich HD Meyer, Robert Loewe

14:00 Quantitative Immuno-PCR based on Imperacer:Technology, Platform & Analytical ApplicationsJan Detmers, Mark Spengler, Michael AdlerChimera Biotec GmbH, Germany

14:30 Quantitative Assessment of Chromatin Structure:Analysis of Epigenetically Regulated Gene PromotersFrancisco BizouarnBio-Rad Laboratories Inc, United States of America

15:00 Quantification of specific mutation with HRM forregions of interestRobert LoeweGeneWake GmbH, Germany

15:30 Quantitation of Rare Undifferentiated hESCs byMethylation-Specific CastPCR (MeS castPCR)Jason Gioia, David Deng, Jeff Rosner, Caifu ChenLife Technologies, United States of America


16:30 PrimeTime® Pre-designed qPCR Assays using ZEN™Internal Dark Quencher ChemistryScott D. RoseIntegrated DNA Technologies, United States of America;

17:00 PrimerXL: high-throughput assay design for qPCRand amplicon based NGSFilip Pattyn, Steve Lefever, Frank Speleman, JoVandesompeleGhent University, Belgium

17:30 Hot Start dNTPs – Novel Chemistries for Use inAdvanced PCR ApplicationsNatasha PaulTriLink BioTechnologies, Inc., United States of America

17:45 PCR-Luminex Based Detection of 28 EnteropathogensMami Taniuchi1, Jie Liu1, Shihab Sobuz2, William Petri1,Rashidul Haque2, Eric Houpt1

1University of Virginia, United States of America;2International Centre for Diarrhoeal Diseases andResearch, Bangladesh

18:00 Simple analytical and experimental procedure forselection of reference genes for qPCR normalizationdataRodrigo Manjarin1, Nathalie Trottier1, Patty Weber2, JamesLiesman1, Nathanael Taylor1, Juan Pedro Steibel1,3

1Department of Animal Science, Michigan State University,United States of America; 2Department of Large AnimalClinical Sciences, Michigan State University, United Statesof America; 3Department of Fisheries and Wildlife,Michigan State University, USA

19:00 – 24:00 Symposium Gala Dinner

Location: Lindenkeller Pasta & More, Freising

International Buffet and Asian Buffet

Music and Dancing


Wednesday 30th March 2011

Pre-PCR: Pre-analytical Steps

Time: Wednesday, 30/03/2011: 9:00am - 12:00pmLocation: Lecture hall 14Session Chair: Jo Vandesompele

Karl H. Hasenstein

9:00 Measurable impact of RNA quality on gene expressionresults from quantitative PCRJoëlle Vermeulen1, Katleen De Preter1, Steve Lefever1,Justine Nuytens1, Fanny De Vloed1, Stefaan Derveaux1,Jan Hellemans2, Frank Speleman1, Jo Vandesompele2

1Ghent University, Ghent, Belgium; 2Biogazelle,Zwijnaarde, Belgium & Ghent University, Ghent, Belgium

9:30 Isolation and Analysis of Circulating Nucleic Acids:Technical AdvancesMartin HorlitzQIAGEN GmbH, Germany;

10:00 Solid Phase Gene Extraction – Sampling of mRNAfrom living systemsKarl H. Hasenstein, Min ChenUniv. Louisiana Lafayette, United States of America


10:30 Impact of basepair mismatch in primer annealing onqPCR assay performanceSteve Lefever, Pattyn Filip, Speleman Frank, HellemansJan, Vandesompele JoCenter for Medical Genetics Ghent, Ghent University,Belgium

11:00 Gene Expression profiles from FFPE samples withimproved RNA decrosslinking technology A casestudy: Molecular profiling of breast cancer fromformalin-fixed, archival materialGuido Krupp1, Susanne Quabius2, Rolf Jaggi31AmpTec GmbH, Hamburg, Germany; 2Institute forImmunology, University Klinikum UK-SH, Kiel, Germany;3Dept. Clinical Research, University of Bern, Bern,Switzerland

11:30 DEPArray™: a novel platform to identify, isolate andcollect very rare cellsPim van der Aar1, Manuela Banzi21Dync B.V., Netherlands, The; 2Silicon Biosystems SpA;



New qPCR Applications in Molecular Diagnostics

Time: Wednesday, 30/03/2011: 13:45pm - 4:30pmLocation: Lecture hall 14Session Chair: Andreas Nitsche

Heinrich HD Meyer

13:45 Detection and classification of microorganisms bycombination of qPCR and pyrosequencingKati Schroeder, Andreas NitscheRobert Koch-Institut, Germany

14:00 FRET primer for single nucleotide polymorphism(SNP) genotypingChoy Len Fong1, Fiona Ng1, Kanaga Sabapathy2, KimHalpin1

1Life Technologies, Singapore; 2Laboratory of MolecularCarcinogenesis, National Cancer Centre, Singapore;

14:15 qPCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2 (AAV2) ITR-SequencesChristine Aurnhammer, Maren Haase, Anja Ehrhard,Martin Häusel, Ingrid Huber, Nadine Muether, HansNitschko, Ulrich Busch, Andreas Sing, Armin BaikerBavarian Health and Food Safety Authority (LGL),Germany

14:30 Cost-efficient detection of Mycobacterium aviumsubsp. paratuberculosis in faeces by quantitative realtime PCR and development of a predictive model forfighting the diseasePetr Kralik1, Iva Slana1, Alena Kralova1, Vladimir Babak1,Robert H. Whitlock2, Ivo Pavlik1

1Veterinary Research Institute, Czech Republic;2University of Pennsylvania, School of VeterinaryMedicine, PA, USA


15:15 Development of a novel duplex real-time PCR assayfor plasma DNA quantification and its comparison toother methodsShiyang Pan, Dan Chen, Peijun Huang, Bing Gu, FangWang, Jian Xu, Chun Zhao, Yongqian Shu, Di YangThe first affiliated Hospital with Nanjing Medical University,China

15:30 Autocrine and paracrine role of leptin andthrombopietin in controlling ovarian function in cowMihir Sarkar1,2, S Schilffarth1, D Schams1, HHD Meyer1, MW Pfaffl1, S Ulbrich1, B Berisha1,3

1Technical University Munich, Germany; 2Physiology &Climatology, Indian Veterinary Research Institute,Izatnagar, Bareilly, Uttar Pradesh, India; 3Faculty ofAgriculture and Veterinary, University of Prishtina,Prishtine, Kosovo

15:45 ULTRA-RAPID REAL-TIME PCR for the detection ofviral diseases in honeybeeByoungSu Yoon, MiSun Yoo, JiNa No, Van Phu NguyenKyonggi University, South Korea

16:00 Development of quantitative triplex real time PCR forthe simultaneous detection of Mycobacterium aviumsubsp. avium and M. a. subsp. hominissuisIva Slana, Maria Kaevska, Petr Kralik, Alice Horvathova,Ivo PavlikVeterinary Research Institute, Czech Republic

16:15 Rapid KRAS, EGFR, BRAF and PIK3CA MutationAnalysis of Fine Needle Aspirates using allele-specificqPCRRonald van Eijk, Jappe Licht, Hans Morreau, Tom vanWezelLeiden University Medical Center, The Netherlands

Closing of the SymposiumLecture hall HS 14

16:30 Closing of the SymposiumHeinrich HD. Meyer & Michael W. Pfaffl


Data Analysis: qPCR BioStatistics &BioInformatics

Time: Wednesday, 30/03/2011: 9:00am - 12:00pmLocation: Lecture hall 15Session Chair: Michael W Pfaffl, Ales Tichopad

9:00 Interpretation requires context - making sense out ofgene lists and networksPhilip ZimmermannETH Zürich, Switzerland

9:30 Stratified error in the qPCR assays from the statisticalpoint of viewAles TichopadAcademy of Science of Czech Republic, Czech Republic

10:00 Primer3: Improvements for the design of qPCRprimersAndreas Untergasser1, Ioana Cutcutache2, Steve Rozen2

1University of Heidelberg, Germany; 2Duke-NUS GraduateMedical School, Singapore


11:00 Is normalisation of raw data necessary? TrulyAutomated Analysis of qPCR Data Using theAzurePCR MethodDavid Kennard, Ze'ev RussakAzure PCR Limited, United Kingdom

11:20 Amplification efficiency as a function of primer andcDNA concentrationJan M Ruijter1, Quinn D Gunst1, Peter Lorenz2, Maurice JBvandenHoff1

1Academic Medical Center, Amsterdam, Netherlands, The;2University of Rostock, Rostock, Germany

11:40 Evaluation and applicability of advanced/exotic qPCRquantification strategies and their implementationAndrej-Nikolai SpiessUniversity Hospital Hamburg-Eppendorf, Germany

12:00 High-resolution melting data error evaluation betweenruns.Maksim Bratchikov, Mykolas MauricasCentre for Innovative Medicine, State Research Institute,Vilnius, Lithuania



GENEX: data analysis lunch seminar

Time: Wednesday, 30/03/2011: 1:00pm - 2:00pmLocation: Lecture hall 15Session Chair: Mikael Kubista

Amin Forootan

13:00 Presentation of GenEx 5.3; the most user friendly andpowerful qPCR experimental design, data analysisand data mining software yet released!Mikael KubistaTATAA BIOCENTER, Sweden

MIQE: state of the art & open discussion

Time: Wednesday, 30/03/2011: 2:00pm - 3:00pmLocation: Lecture hall 15Session Chair: Stephen Andrew Bustin

Jo Vandesompele


RDML: state of the art & open discussion

Time: Wednesday, 30/03/2011: 3:30pm – 4:30pmLocation: Lecture hall 15Session Chair: Jan Hellemans

Andreas Untergasser

Closing of the SymposiumLecture hall HS 14

16:30 Closing of the SymposiumHeinrich HD. Meyer & Michael W. Pfaffl


Thursday 31st March & Friday 1st April 2011

qPCR Application Workshops

The workshops are aimed at giving participants a deep and objective understanding of real-time quantitative PCR, biostatistics, expressionprofiling, and its applications. The courses are intended for academic or industrial persons considering working with qPCR or scientists currentlyworking with qPCR seeking a deeper understanding.

The qPCR courses cover all aspects in qPCR and are held during 2-days. Each course is approximately 50% hands-on and is limited to 20-25participants (biostatistics 40-50 participants), resulting in very interactive teaching and everybody given the opportunity to try theinstrumentation. After the course participants will be able to plan and perform qPCR experiments themselves, as well as interpret and analyzedata. Detailed course material and full catering (lunch, coffee, soft drinks and snacks) are included in the course fee.

All workshops start on Thursday and Friday at 9 am until 5 pm. All three workshops are hosted by the TATAA Biocenter Sweden, and bioEPSGmbH (www.tataa.com www.bioeps.com). The qPCR workshop laboratories and seminar rooms are close to the central lecture hall.

Workshop topics:

Basic Module qPCR Application Workshop (2-days) Practical room – P1

qPCR Biostatistics & Expression Profiling Workshop (2-days) Computer seminar room – PU

MIQE guidelines (1 day) & Practical primer design (1-day) Seminar room - S1

Basic Module qPCR Application Workshop (2-days) Practical room – P1

The introductory course consists of a theoretical part and a practical part where participants get to do QPCR experiments by themselves underexperienced supervision. The course contains:

Day 1 - hands on workshop:• Basic PCR theory• The theory of real-time PCR• Applications and possibilities of qPCR. Comparison of qPCR with regular PCR.• Review of currently available detection technologies (SYBR Green I, hydrolysis probes, Molecular Beacons...etc)• Different instrument platforms and their typical uses• Primer design• The problem of primer-dimer formation and how to minimize them• Probe design• Experimental design and optimization• Basic data handling and analysis

Day 2 - hands on workshop:• Introduction to quantification principles• Quantification strategies, uses and limitations• Calculations using different relative quantification methods• Strategies for normalization of qPCR data• In situ calibration for compensation of inhibition in samples• Absolute quantification

qPCR data analysis - Biostatistics & Expression Profiling (2-days) Computer seminar room – PU

This course will teach you how appropriate statistics should be selected and applied correctly to get the most out of your qPCR data. The coursemixes theoretical lectures with computer based exercises. Please bring your own Laptop to the course!

Day 1- Statistical analysis of real-time PCR data• Basic principles of statistics• Advanced principles of statistics• Statistical tests• Ability to detect a difference

Day 2- Gene expression profiling with real-time PCR• Multiplate measurements• Standard curves and absolute quantification• Experimental design, selecting reference genes• Relative quantification, comparison of groups• Expression profiling


MIQE guidelines (1 day) & Practical primer design (1-day) Seminar room - S1

Day 1 - The MIQE guidelines – How to improve your qPCR quality

This is a theoretical course that will take you through the MIQE guidelines. The MIQE guidelines (Minimum Information for Publication ofQuantitative Real-Time PCR Experiments) have been developed to ensure the integrity of the scientific literature, promote consistency betweenlaboratories and increase experimental transparency. The course will explain the different steps that are necessary to fulfill to ensure goodqPCR quality and show you how it can be done. The course includes:

• Introduction to the MIQE guidelines• Sample preparation• Nucleic acid extraction and quality control• Reverse Transcription• Primer and probe design• Optimization and validation of qPCR• Normalization• qPCR data analysis

Day 2 - Practical primer design

This is an extensive primer design course that will teach you how to design your own assays. It will teach you the theory of primer and probedesign as well as how to optimize your reactions. The course will also let you do design of your own primers using primer design softwaresunder experienced supervision.

qPCR Workshop Sponsors:


Industrial Exhibition

35 companies participate at the industrial exhibition held during the qPCR Symposium March 28th – 30th in the foyer of the central lecture hallcomplex (green frame) and in two side rooms S1 and S2 (blue frame).

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Registration & Catering

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1&2 Agilent Technologies 18 Life Technologies

3 Genewave 19 Fujifilm

4 Bio-Rad 20 MMI

5 Qiagen 21 Eurogentec

6 Nanostring 22 Lonza

7 Roche Applied Science 23 Sigma Life Science

8 Thermo Fisher Scientific 24 Amplifa

9 Biosearch Technologies 25 Fluidigm

10 Exiqon 26 Biolegio

11 Eurofins MWG Operon 27 TATAA & MultiD

12 TaKaRa Bio Europe 28 Wafergen

13 Metabion 29 ArcticZymes

14 Bioke 30 Biogazelle

15 TIB Molbiol 31 Analytik Jena & Biometra

16 Integrated DNA Technologies 32 Primerdesign

17 Bioline 33 Affymetrix

17 4titude 34 Illumina

RestRooms

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Abstracts - Oral presentations

Monday 28th

March 2011

MIQE and QC strategies in qPCR

Time: Monday, 28/03/2011: 10:30am - 1:00pmLocation: Lecture hall 14Session Chair: Stephen Andrew Bustin, Michael W Pfaffl

MIQE: compliance and amendments

Stephen Andrew Bustin1, Jo Vandesompele2

1Barts and the London School of Medicine, QMUL, UnitedKingdom; 2Center for Medical Genetics Ghent (CMGG) GhentUniversity Hospital

The publication of the minimum information for the publication ofreal-time quantitative PCR experiments (MIQE) guidelines hasbecome a defining event in the maturing of qPCR technology. Theresponse from instrument and reagent manufacturers has beenuniversally positive; there has been extensive publicity in print,online and at scientific meetings and many scientific journals arebeginning to take note. We report the results of the first extensivesurvey carried out to determine transparency and quality of recentqPCR publications. It has also become apparent that an importantobstacle to the universal acceptance of the MIQE guidelines hasbeen the stipulation for primer sequence disclosure. Manycommercial qPCR assays do not provide this information; usuallythere are also no details provided on empirical validation of eachindividual assay. The increasing use of commercial qPCR assaysis creating problems, as it results in publications that cannotsatisfy current MIQE requirements. Consequently, we propose anamendment of the original guidelines to help users of commercialassays publish transparent protocols that fulfil the MIQE criteria.

Convenient and reliable gene expression profilingusing RealTime ready Focus Panels in combinationwith sound data-analysis using qbasePLUS

Ali Rihani1, Tom Van Maerken1, Barbara D'haene2, NurtenYigit1, Jo Vandesompele1,2, Rita Rein3

1Center for Medical Genetics, Ghent University, Ghent, Belgium;2Biogazelle, Ghent, Belgium; 3Roche Diagnostics, Germany

Roche’s RealTime ready Focus Panels are ready-to-use, pre-validated qPCR assays targeting selected genes from specificpathways or functional groups. We aimed to evaluate the utility ofthe technology for convenient and reliable gene expressionanalysis in accordance to the MIQE guidelines. A pilot experimentwas set up using the human apoptosis panel that enablesexpression profiling of 372 genes. Two previously establishedchemosensitive and chemoresistant neuroblastoma cell lines wereanalyzed before and after treatment with the MDM2 inhibitornutlin-3. Thorough post-qPCR quality control and data-processingwas performed using Biogazelle’s qbasePLUS software. We wereable to demonstrate (1) the expression stability of a selectedsubset of the included reference genes, (2) the utility of theintegrated positive and negative RT controls and RNA integrityassessment, (4) the ease and power of data analysis usingqbasePLUS, and (3) the power of the strategy to obtain biologicallyrelevant results when scrutinizing an entire pathway. Overall, thestudy demonstrates that the RealTime ready Focus Panels incombination with the state-of-the-art real-time PCR data-analysissoftware qbasePLUS enable easy, fast, and reliable geneexpression quantification.

"Stay in touch while on the bench" MIQE qPCR

Afif Abdel Nour1, Michael W. Pfaffl2

1LaSalle Beauvais, France; 2Physiology Weihenstephan, TUM,Germany

Who does not know iPhone and iPad devises? More and morepeople have an Apple mobile device such as iPhone, iPad or iPodtouch. From now on, those mobile devices can be used to

increase quality of qPCR experimant or publication, by providing a'MIQE APP'. This new application could be used by scientists tocheck whether their experiment or the used literature fulfills theMIQE requirement or not. The 'MIQE Guidelines' checklistprovides 85 parameters that qPCR studies should be required orrecommended to meet before being considered for publication.This checklist is based on the original published MIQE checklistand we hope it will increase future publication quality andreliability. But there are much more wider seen goals of the 'MIQEguidelines', all in all the goals might be summarized; to increasereliability of results to help to insure the integrity of scientific work,with major focus on biological relevance. The MIQE applet willprovide an interactive and portable follow up of these parametersfor any qPCR research application. The applet will be free fordownload and upon installed connected to our MIQE databank,collecting all the information. The 'MIQE guidelines' (MinimumInformation for Publication of Quantitative Real-Time PCRExperiments) were published by a group of qPCR experts in 2009(Bustin et al, Clinical Chemistry 2009).

qPCR Gene Expression Assays and an ExogenousRNA control for indication of reaction inhibition in theRT-qPCR workflow

Jaakko Kurkela

Thermo Fisher, Finland

Reverse transcriptase quantitative PCR (RT-qPCR) is a routinelaboratory technique for the measurement of RNA that is bothsensitive and specific with appropriate assay design. ThermoScientific Solaris qPCR Gene Expression Assays are probe-basedassays that are ideal for routine molecular applications; theycombine minor groove binder technology (MGB) with a rigorousalgorithm that permits detection of all known splice variants of agene target, while distinguishing among closely related familymembers, on a genome-wide scale. Inhibitors of RT-qPCR maybe present in experiments and not easily recognized. The primarychallenge is that methods such as spectrophotometric techniquesdo not always detect RT-qPCR inhibitors. The Thermo ScientificSolaris RNA Spike Control Kit was designed as an exogenouscontrol to identify the presence of reaction inhibition includingthose commonly carried through processing steps to isolate RNAsuch as EDTA, phenol, heparin and EtOH. Here we describeapplications for this exogenous control to identify the presence ofreaction inhibition and illustrate the impact inhibition can have onresults and data interpretation. Experimental results demonstrateRNA Spike Control provides accurate identification of RT-qPCRinhibition and the ability to predict when inhibition has beenremoved, for example, by dilution of the RNA sample into thereverse transcription reaction. The RNA Spike Control identifiedRT-qPCR inhibition in a gene expression assay, explaining thevariable results obtained. Furthermore, the use of RNA SpikeControl Kit was compared to current inhibition identificationprotocols and was found to be an easy and succinct workflow,offering a clear advantage of this technology.

Quality control and management approaches forMolecular Diagnostics techniques used duringdiagnosis and monitoring of hematologicmalignancies

Barbara Zehentner

HematoLogics Inc, United States of America

This presentation will provide an introduction to currentapplications of molecular analysis tools in the clinical fieldof hematologic malignancies. Several different assay platforms willbe covered, from B- and T-cell clonality assessment by generearrangement PCR, point mutation analysis for myeloidneoplasms, Sanger sequence analysis for CLL prognosis andGleevec resistance assessment, in addition to the application ofreal-time PCR for quantitative monitoring purposes and minimalresidual disease detection. Quality control and managementapproaches for the clinical reference laboratory will be discussed.


Substantial performance discrepancies betweencommercially available kits for rt-qPCR - A systematiccomparative investigator-driven approach

Matthias W Sieber1,2, Peter Recknagel2, Florian Glaser2, OttoW. Witte1, Michael Bauer2, Ralf A. Claus2, Christiane Frahm1

1Hans Berger Clinic for Neurology, Jena University Hospital,Germany; 2Anaesthesiology and Intensive Care Medicine, JenaUniversity Hospital, Germany

Reverse transcription followed by quantitative PCR (rt-qPCR) hasbecome the state of the art tool forquantification of nucleic acids.However, there are still significant problems associated with itssensitivity, reproducibility, efficiency and the choice of anappropriate rt-qPCR kit. The purpose of this study is to giveinsights into strategies to optimize and validate the performance ofcurrently available kits for rt-qPCR and to provide up-to-dateinformation about the benefits, potentials and pitfalls of rt-qPCRassays. A selection of 9 cDNA synthesis and 12 qPCR kits weretested using samples obtained from three species (mouse, rat andhuman) and three transcripts (Gapdh, Actb and Hmbs) underhighly standardized conditions. Kits with an outstandingperformance were further analysed to identify the dynamic rangefor a reliable quantification of mRNA. Reverse transcriptionefficiency varied up to 90 fold depending on the choice of reversetranscriptase, priming strategy and assay volume. The qPCR kittest revealed variations in mean relative amplification efficiencyranging from 54% to 171%. We conclude that currently availablekits for rt-qPCR vary considerably. However, with an appropriatevalidation strategy and knowledge about capabilities of a particularkit, sensitivity, efficiency and reliability could be significantlyimproved.

single-cell qPCR

Time: Monday, 28/03/2011: 2:00pm - 6:00pmLocation: Lecture hall 14Session Chair: Mikael Kubista, Anders Ståhlberg

25 years of PCR - from idea to subcellular expressionprofiling

Mikael Kubista

TATAA BIOCENTER, Sweden

PCR was born from a great idea conceived by Kary Mullis andlater refined into qPCR by Russ Higushi. Specificity was added byKen Livak. The technique found immediate use in geneticengineering leading to several breakthrough innovations, and laterPCR became the preferred platform for clinical diagnostics. Thequantitative aspect of qPCR became particularly valuable inbiological and medical research, and is today key technology insystems biology. Moreover, the extreme sensitivity of qPCR,allowing the detection of single molecular copies, and the relativeease of use, which allows qPCR to be integrated in a streamlinedhigh throughput workflow, has led to the exciting area of single cellexpression profiling. An astonishing heterogeneity in transcriptlevels among seemingly like cells has been found, whilecorrelation between genes’ expression is a fingerprint of the typeof cell. Most recently, qPCR tomography has been developed tomeasure intracellular mRNA profiles, revealing gradients oftranscripts within the cell, preparing it for asymmetric cell division.

Anders Stahlberg, Daniel Andersson, Johan Aurelius, Maryam Faiz,Marcela Pekna, Mikael Kubista, Milos Pekny. Defining cell populations withsingle-cell gene expression profiling: correlations and identification ofastrocyte subpopulations. Nucl. Acids Res. 1–12. doi:10.1093/nar/gkq1182(2010).

Radek Sindelka, Monika Sidova, David Svec, Mikael Kubista. Spatialexpression profiles in the Xenopus laevis oocytes measured with qPCRtomography. Methods 51:87-91(2010).

M. Bengtsson, A. Ståhlberg, P. Rorsman, and M. Kubista Gene expressionprofiling in single cells from the pancreatic islets of Langerhans revealslognormal distribution of mRNA levels. Genome Research 15, 1388-1392(2005).

Research Highlights in Nature Review Genetics 6, 1758 (2005).

Uncovering the Diversity of Individual Cells: GeneExpression Profiling with the BioMark System

Ken Livak

Fluidigm Corporation, United States of America

Fluctuations in gene expression at the single cell level could bekey for generating developmental signals and for understandingthe progression of tumors. Data needs to be collected from astatistically significant number of single cells in order to determinethe range of gene expression present in a population of cells.Furthermore, transcripts need to be quantified for a number ofgenes in order to obtain meaningful cell signatures. BioMark™arrays provide a convenient and cost-effective system forperforming multiple RNA expression assays on multiple single-cellsamples. This system has been used to study single cell geneexpression in embryonic stem cells, hematopoieticstem cells,cancer stem cells, and early stage embryos.

Identification of unknown cell populations andcorrelations using single-cell gene expressionprofiling

Anders Ståhlberg

University of Gothenburg, Sweden

Single-cell gene expression levels show substantial variationsamong cells in seemingly homogenous populations. Access tofundamental information about cellular mechanism, such ascorrelated gene expression, motivates studies of multiple genesexpressions in individual cells. Astrocytes perform many controland regulatory functions in the central nervous system. In contrastto neurons, we have limited knowledge about functional diversityof astrocytes and its molecular basis. We will show howsubpopulations of cells can be identified at single-cell level usingunsupervised algorithms and that gene correlations can be usedto identify differences in activity of important transcriptionalpathways. We identified two subpopulations of astrocytes withdistinct gene expression profiles. One had an expression profilevery similar to that of neurosphere cells, whereasthe other showed characteristics of activated astrocytes in vivo.Technical considerations related to reproducible and efficientsampling, lysis, reverse transcription and real-time PCR will alsobe presented. In addition to astrocytes, single-cell data from tumorcells, beta-cells and embryonic stem cells will be shown.

Single Molecule RNA FISH: novel, simple, andaccurate quantitative applications for gene expressionanalysis

Arjun Raj

University of Pennsylvania, United States of America

We have developed a simple, direct method for specificallydetecting individual RNA molecules in situ via fluorescencemicroscopy. Our RNA-FISH method relies on using large numbersof fluorescently labeled oligonucleotides designed to targetparticular RNA species. This approach is highly specific and highlysensitive and provides absolute, direct quantification of RNAabundance in single cells. Moreover, it is applicable to a broadrange of biological sample types, ranging from microbes to humantissue section. We will discuss the method itself as well asaddress some biological applications in development and cancer.

Relationship between cell attachment and bcr-abl cellexpression heterogeneity within CML cell lines.

Philip Day, Ehsan Karimiani

University of Manchester, United Kingdom

Chronic myelogenous leukemia (CML) is believed to occur as aconsequence of the clonal expansion of leukemic stem cells andto be maintained by an expanding population of hematopoieticstem cells that have acquired a BCR-ABL fusion gene. Recentstudies indicate that primitive CML cells are less responsive totyrosine kinase inhibitors and are a reservoir for the emergence oftyrosine kinase resistant subclones. Some studies havesuggested that expression of BCR-ABL may also be required foraltered cell adhesion and CML progression. The bcr-abl proteinmodulates cell adhesion and its effects in cell lines like K562correlate with increased adhesion to fibronectin. It also has beenreported that cell adhesion mediated resistance to apoptosis


induced by BCR-ABL inhibitors, suggests that bcr-abl mediatedcell adhesion may be involved in post-therapy residual disease ofCML. However, some reports imply that a relatively small fraction(2%–20%) of blasts from patients with acute myeloid leukemiaadhere to the plastic of the cell-culture dish. In this study we test ifelevated BCR-ABL expression could be specifically identified inindividual cells from attached cell populations and not unattachedpopulations. The study developed a means to utilise flow assistedcell sorting of cell lines expressing BCR-ABL to derive individualattached and unattached cell sub-populations. Using ahomogenous extraction procedure, cells individually flow sortedinto microtitre plates were subjected to combinations of abl andbcr-abl qRT-PCR, and revealed the existence of low and high bcr-abl expressing cell line populations. The implications of this studywill be discussed.

Digital Competitive Allele-Specific TaqMan-BasedReverse Transcription-qPCR (castRT-qPCR) for DirectDetection and Enumeration of Circulating Tumor Cells(CTCs)

David Xingfei Deng, Yun Bao, Scott Sproul, Yu Wang, FawnWang, David Merrill, Pius Brzoska, Caifu Chen

Life Technologies, United States of America

Molecular characterization and enumeration of circulating tumorcells (CTCs) promise to be valuable for cancer cell diagnosis,survival prognosis, and treatment guidance. However, currentmolecular assays require extensive blood sample enrichmentprocess before analyzing extraordinarily rare CTCs. Here wereported a new approach for direct CTC molecular detection inwhole blood samples without prior biophysical processing bycombining sample partition and digital assay of a highly specificcastRT-qPCR. CastPCR can detect rare copies of mutant alleleswith a wide dynamic range of more than 6-log orders and < 5-copysensitivity. Whole blood samples with spiked-in known mutationlung cancer cell lines and from lung cancer patients werepartitioned in aliquots of 2.5 μL – 50 μL onto 96- or 384-wellplate(s), such that each well contains either one cancer cell ornone with per well 20,000 – 400,000 normal white blood cells and10 – 200 million red blood cells. RNAs and/or DNAs wereextracted by magnetic beads and directly or were pre-amplifiedprior to mutation detection. Genetic mutations and cell typespecific markers (such as CK19 and/or CEA) for CTC identificationand enumeration were determined by castRT-qPCR and/orTaqMan Gene expression assays. The sample partition processresulted in a relative CTC enrichment or digital enrichment of 20 –400 folds (the relative ratio of CTC to normal cells) in a CTC-positive well. CastPCR clearly identified known mutation andCK19 in spiked-in samples of about 10 – 30 cells per mL wholeblood, but detected no mutation signals in any sample well withoutcell spiked-in. Furthermore, cell type specific markers (CK19) andknown EGFR mutation(s) were identified in the same samplewells, suggesting identified mutation is specific and from cancercells not from normal cells. In five blood samples from lung cancerpatients, EGFR mutation (p.L858R) was detected in all samples.Approximately 50% of circulating lung tumor cells in a patient withpositive EGFR p.L858R mutation had also positive EGFRp.T790M mutation, an inducible drug-resistant CTC marker. Forthose samples with negative detection of EGFR mutation,corresponding wild type sequences were detected in all samplewells, suggesting the normal DNA amplification of those mutation-negative wells. Our data suggest that combination of digitalsample enrichment and castRT-qPCR can be used to directlyenumerate CTCs and detect cancer-related mutations in wholeblood without prior biophysical sample enrichment. The newapproach paves way for noninvasively CTC monitoring andindividualized therapy.

Real time PCR on slide: a new tool for quantification ofminute amounts of DNA

Jean-Christophe Avarre1, Angelique le Bras2, Régis Melizzi2,Maxime Rattier2, Gordana Cerovic2, Claude Weisbuch2,Marianna Alunni3, Martin Kantlehner3, Petra Hartmann3,Reinhard Bittner3, Wolfgang Mann3

1Institut de Recherche pour le Développement, Montpellier,France; 2Genewave SAS, Paris, France; 3Beckman CoulterBiomedical GmbH, Munich, Germany

Detection, identification and quantification of micro-organismsrepresents one of the major challenges for our modern society. Itconcerns a wide variety of applications including bioprocesscontrol, food technology, health care, environmental analysis andof course clinical diagnostics. In this regard, miniaturization ofPCR protocols may offer many advantages including short assaytime, high precision and sensitivity, high-throughput, low reagentconsumption and portability. Though on-chip PCR is likely tobecome the 'next-generation PCR', translating microfluidics tobiology labs is still limited by its cost and the expertise it requires.In this context, a new format system has been developed, whichallows DNA extraction and real-time PCR amplification in 1-µl"reaction sites" on a slide. The system, called SicLive, enables toprocess 48 samples in parallel and offers the possibility to performquantitative PCR amplifications within 2 hours, including DNAextraction. Results, obtained on different model systems, showedthat reproducibility and dynamic range were comparable to thoseobtained in 20 µl with conventional real-time PCR. In terms ofsensitivity, SicLive was able to amplify DNA from 1-5 copies oftarget as well as from single cells. By combining DNA extractionand amplification in a single reaction site, SicLive is particularlywell adapted for quantifying precious samples containing very lowamounts of genetic material, such as clinical or environmentalsamples. Finally, for single-cell applications, this slide formatensures an easy visual control, with standard microscopy, of bothquality and quantity of the templates loaded.

Towards a Comprehensive Single Cell ExpressionProfiling

Herbert Auer

IRB Barcelona, Spain

Expression profiling, the measurement of all transcripts of a cell, iscurrently the most comprehensive method to describe itsphysiological state. Given that accurate profiling methods currentlyavailable require RNA amounts found in thousands to millions ofcells, many fields of biology working with specialized cell typescannot use these techniques because available cell numbers arelimited. Currently available alternative methods for expressionprofiling from picograms of RNA or from very small cellpopulations lack a broad validation of results to provide accurateinformation about the measured transcripts. Except for a fewhighly experimental methods like Single-Molecule-Sequencing,every attempted measurement of more than a few differenttranscripts needs a pre-amplification of cDNA. Here we presentPico Amplification, a novel workflow of RNA isolation and cDNAamplification for cell populations as small as a few or even a singlecell. Micrograms of cDNA are generated, suitable for analysis byqPCR, microarrays or sequencing. Expression profiling of RNAsdiluted to picograms showed that Pico Amplification virtually didnot alter measurements of differential expression. MAQC samplesA and B (Universal Reference RNA and Human Brain RNA) wereutilized to provide transcriptome wide evaluation using microarraysand for comparison to qPCR measurements for over 800transcripts. RNA isolated from 10 cells provided after PicoAmplification virtually identical information about the entirepopulation as standard protocols do from thousands or millions ofcells. This held true across the entire transcriptome. RNA isolatedfrom individual cells provided insight into the heterogeneity ofseemingly homogenous cell populations. Hundreds of transcriptswere found to be differentially expressed between individual cells.Pico Amplification provides micrograms of cDNA from very smallcell populations or even individual cells, highly representative ofthe original transcriptome. This allows the application of virtuallyall standard methods of expression profiling to interrogate thetranscriptional status of an individual cell.


Tuesday 29th March 2011

High throughput & digital PCR

Time: Tuesday, 29/03/2011: 8:30am - 12:00pmLocation: Lecture hall 14Session Chair: Frank McCaughan

Kevin L Knudtson

Using Nanoscale PCR and NGS Technologies toDetect Rare Sequence Variants in a Core Setting

Kevin L Knudtson

University of Iowa, United States of America

The University of Iowa DNA Facility has been providing sequencevariant detection services using nanoscale PCR and NGStechnologies for nearly three years. Detection of sequencevariants that occur in a small percentage of the population has metwith both technical and analytical challenges in avoiding orminimizing false calls. False positive calls can be very expensiveto verify. False negative calls simply cannot happen as they resultin a missed diagnosis. Using the Applied Biosystems OpenArrayand Fluidigm Dynamic arrays, the ability of each platform to givethe correct genotype for 32 assays over 575 individuals wasevaluated. Both platforms were similar in their false call rate andthe correct call rates improved with by conducting a specific targetenhancement of the samples prior to the genotyping assay.Variant detection was also evaluated using NGS technologies.Amplicons from 300 individuals interrogating over 210 targetregions from each individual were mixed together and run over theRoche GS FLX and Applied Biosystems SOLiD genomesequencers. False positive rates were initially high, but decreasedas the specificity of the primers was improved. Workflows and/orexperimental design enhancements to reduce false genotypingcall rates will be discussed.

Customized, function tested RealTime ready assaysfor gene expression analysis in biomarker researchand early drug development

Sabine Lohmann1, Andrea Herold1, Bergauer Tobias3,Belousov Anton2, Betzl Gisela1, Dietrich Manuel1, HesselHarald5, Poignee-Heger Manuela1, Geho David4, WeisserMartin2

1Roche Applied Science, Penzberg, Germany; 2Roche Pharma,Penzberg, Germany; 3Roche Pharma, Basel, Switzerland; 4RochePharma, Nutley, USA; 5Ludwig-Maximillian-University, Institue ofPathology, Munich, Germany

Personalized Health care (PHC) uses gene-based information tounderstand requirements for health maintenance, diseaseprevention, and therapy, all tailored to an individual’s geneticuniqueness. Biomarkers are therefore a central elementthroughout a drug’s lifecycle, from target identification to drugapplication. In biomarker research and early drug development,the investigation of mRNA expression profiles is of great interest.Roche RealTime ready custom panels offer a broad variety ofintron-spanning RT-qPCR assays covering the major signaltransduction pathways, including those relevant for oncologyresearch. RealTime ready custom panels are comprised of ready-to-use LightCycler® 480 Multiwell Plates containing pre-platedqPCR assays for human, mouse, and rat targets that have beenselected by the user. We have established various workflowsapplicable for gene expression analysis in biomarker research aswell as in pre-clinical and early clinical studies. Pre-clinical drugdevelopment was started by profiling gene expression using amulti-parameter panel (93 target and 3 housekeeping genes)covering the pathway of interest in a broad screening approach.The goal was to generate a first hypothesis for predictive andpharmacodynamic markers, as well as to provide a workflow withthe highest convenience and throughput for this screeningapproach. This first step was performed in vitro with tumor celllines treated with the compound of interest. A workflow protocolwas developed, starting with an automated RNA extraction on theMagNA Pure LC Instrument in combination with pre-platedRealTime ready customized assays in a 384-well format. Therelative gene expression had been analysed. After selecting a setof parameters, this first hypothesis was verified using an in vivo

mouse model. Xenograft-derived fresh frozen (FF) tissue sampleswere used to test for selected biomarkers. Next, we moved toearly clinical development using the first set of clinical samples,formalin-fixed paraffin-embedded (FFPE) tissue derived fromvarious tumor entities. Tumor-derived FFPE tissue is the mostrelevant sample material for the development of therapeuticcompounds in clinical trials or in biomarker research. RNAextraction from FFPE tissue (e.g., 10 µm sections) using the HighPure RNA Paraffin Kit, followed by qRT-PCR analysis, is a wellestablished technique; here, we combined this workflow withfunction tested RTR assays.

For life science research only. Not for use in diagnostic procedures.

LIGHTCYCLER, MAGNA PURE, HIGH PURE, and REALTIME ready aretrademarks of Roche.

Digital PCR - getting the most out of difficult clinicalsamples

Frank McCaughan

Medical Research Council, United Kingdom

We use digital PCR to interrogate clinical biopsy samples both forbasic research and translational medicine purposes. For this weuse a technique called Molecular Copy-number Counting which isessentially a multiplex hemi-nested single molecule digital PCRprotocol. The advantages of MCC are that we can use it to extractmultilocus information from tiny quantities of starting DNAextracted from formalin-fixed paraffin-embedded archivedmaterial. In this talk I will describe the MCC strategy in the generalcontext of digital PCR, how we have applied it to challengingclinical samples and how we have now modified the protocol toderive both multilocus copy-number and sequence information(including using NGS) from these samples.

Digital PCR or Finding The Needle In A Haystack

Elena Grigorenko

Life Technologies, Inc, United States of America

Digital PCR is a new approach to nucleic acid detection andquantification, which allows to perform absolute quantification oftargets by directly counting the number of target moleculespartitioned into many individual real-time PCR reactions. Theintroduction of OpenArray® Real-Time PCR System to performTaqman™ chemistry with digital PCR enable researchers to run9,000 PCR reactions simultaneously. This approach can be usedfor a variety of different applications aimed on detection of rareevents such as quantification of viral load in diluted plasmasamples, rare allele detection and single stem cell transcriptanalysis. The examples of each application will be discussed.OpenArray® platform and its new digital PCR capability not onlyextend the portfolio of conventional PCR applications, but alsoprovide a new, more precise answers to many different biologicalquestions.

Digital Analysis- a bridge to the Clinic

Jim White

NanoString Technologies, United Kingdom

The nCounter System utilizes molecular barcodes and direct singlemolecule imaging to detect and count hundreds of uniquebiomarkers in a single reaction. The nCounter assay directlytargets nucleic acid molecules through hybridization, eliminatingthe need to purify sample, let alone make cDNA or amplify thesignal. The truly digital nature of quantification provides anaccuracy and reproducibility unmatched with other systems. ThenCounter system can work with a large variety of samples, fromRNA purified from fresh biopsies to crude extracts made fromFFPE material. With a portfolio of off-the-shelf and custom-designed applications in mRNA, miRNA and copy numberanalysis, the nCounter system is well suited to a variety ofbiological problems. In our presentation we will show how theCodeSet technology, can construct subtle sample enquires thatcan with simple iteration unlock the complexity in the interfacebetween genome and transcriptome. The simple utility of theSystem will help provide an ideal format to empower molecularmedicine by providing a bridge to take nucleic acid biomarkersfrom research to the Clinic.


High-throughput real-time PCR approach using theLightCycler®1536 instrument for genotyping and highdiscrimination of E. coli pathogroups responsible forgastrointestinal diseases

Sabine Delannoy1, Lothar Beutin2, Cédric Woudstra1, PatrickFach1

1French Agency for Food, Environmental and Occupational Health& Safety (ANSES), France; 2NRL for E. coli, Federal Institute forRisk Assessment, Germany

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens responsible for a number of humangastrointestinal diseases, including watery or bloody diarrhea, andhemorrhagic colitis. Rapid and specific detection of STEC strainswith high virulence for human has become a priority for publichealth authorities. The main virulence factor of STEC is theproduction of Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2) or itsvariants. STEC strains causing severe disease in humans mainlypossess additional virulence factors such as the outer membraneprotein intimin, essential for the intimate attachment and theformation of attaching and effacing (A/E) lesions ongastrointestinal epithelial cells whose genetic determinant isharboured by the LEE pathogenicity island. In addition to the LEElocus, several other putative pathogenicity islands (PAIs) havebeen identified in STEC strains which present a variable repertoireof effectors genes that encode potential virulence determinants(nle, as non-LEE encoded effector). Based on a high-throughputreal-time PCR approach using the LightCycler®1536 instrument aselection of discriminative nle genetic markers mostly encodingtype III secretion system effector proteins allowed to develop anassay for specific characterization of the most virulent strains ofSTEC. These nle genes were detected at different frequencies inpathogenic and apathogenic E. coli strains indicating their possiblerole in virulence. The presence of these genes may be used tomake a “molecular risk assessment” to predict the virulencepotential of STEC strains. For E. coli O26, these data werecorroborated by a genotyping real-time PCR test based on allelicdiscrimination of the arcA (aerobic respiratory control protein A)gene. Results obtained with the LightCycler®1536 instrumentindicate that a combination of molecular detection and genotypingtools are highly discriminative to clearly distinguish STEC strainsconstituting a severe risk for human health from STEC that are notassociated with severe and epidemic disease. This simplediagnostic approach might be applicable in hospital service labs orpublic health laboratories to test strains isolated from foods orstools of patients suffering from diarrhea.

NGS: Next Generation Sequencing

Time: Tuesday, 29/03/2011: 2:00pm - 6:00pmLocation: Lecture hall 14Session Chair: Yuk Ming Dennis Lo, Yu-Hui Rogers

Noninvasive prenatal diagnosis using fetal DNA inmaternal plasma: from digital PCR to next-generationsequencing

Yuk Ming Dennis Lo

The Chinese University of Hong Kong, Peoples Republic of China

Prenatal diagnosis is now an established part of modern obstetricscare. However, conventional definitive methods for prenataldiagnosis, such as amniocentesis, are typically invasive andconstitute a risk to the fetus. In 1997, our group reported that cell-free fetal DNA is present in the plasma of pregnant women. Thistechnology has initially been used to detect paternally-inheritedfetal genetic characteristics, e.g. sex and blood group genes thatare absent in the mother. In 2007, our group reported the use ofdigital PCR for the precise quantitative analysis of fetal DNA inmaternal plasma. We demonstrated the theoretical and practicalbasis for single molecule analysis for the noninvasive prenataldiagnosis of trisomy 21. We have since demonstrated thatmassively parallel sequencing is a robust method for thenoninvasive detection of fetal trisomy 21 and have recently

reported a large scale study using this technology. Our resultsindicate the fetal trisomy 21 can be detected with a sensitivity of100% and a specificity of 98% using this technology. We havealso recently shown that in addition to trisomy 21, maternalplasma DNA sequencing can also reveal the genome-wide geneticmap of the fetus. Thus, massively parallel sequencing is likely toplay an increasingly important role in the future prenataldiagnostics and monitoring.

Qualitative and Quantitative Analysis of RNA by SOLiDNext Generation Sequencing

Thomas Rygus

Life Technologies GmbH, Germany

The last years have seen Next Generation Sequencing (NGS) asan upcoming new method replacing many of the established arraybased methods, especially in RNA analysis. Users of RNAsequencing published in a variety of scientific fields beginning withquantitative gene expression analysis up to structural analysis ofRNA and allele specific expression; also combinations of severalNGS techniques like small RNA sequencing and ChIP Seq havebeen shown. Meanwhile an established method, quantitative RNAanalysis by SOLiD now allows the use of internal RNA controlsmaking sure that a reliable standardization is possible.The biggest challenges with resequencing methods have beenautomating the complete process and improving the turnaroundtime for smaller amounts of samples without losing cost efficiency.In this presentation we will show an overview about the newesttechnical developments of the SOLiD system and workflowtogether with an overview about the possible applications in RNAanalysis.

Analysis of circulating nucleic acids (CNA) using NGStechnologies

Bertram Brenig1, Julia Beck2, Ekkehard Schütz2, Howard BUrnovitz2

1University of Goettingen, Germany; 2Chronix Biomedical GmbH,Germany

Next generation sequencing technology provides significantpromise in providing personalized medicine in the 21st century. Arecent report has clearly demonstrated in a small number ofpatients that unique chromosomal rearrangements identified bynext generation DNA sequencing can be detected in blood byPCR. Nevertheless significant barriers to successful translation ofthis advancement to clinical medicine exist that include knowledgeof unique mutational lesions for each neoplasm and their fateduring clonal expansion. We have used an alternative approachthat is dependent on next generation sequencing of serum DNAand DNA motif identification and assembly (Mass Sequencing andAssembly or MSA) technology that requires no prior knowledge ofspecific mutational events in diseased tissue. We havesuccessfully applied MSA technology to invasive breast carcinomaand neurodegenerative diseases of animals and humans. Webelieve this alternative approach provides an opportunity fortranslation of complex basic research data to personalizedmedicine.

Improved library quantification for High ThroughputSequencing at the Wellcome Trust of Human GeneticsOxford

Paolo Piazza

Wellcome Trust Centre for Human Genetics, United Kingdom

Recent advancements in sequencing technologies (NextGeneration Sequencing) developed for the Illumina and LifeTechnology platforms have driven down significantly the costs perbase sequenced. Lower costs coupled with significantimprovements in data quality have opened the door to sequencingprojects that had previously been far too expensive to complete.As a result we are witnessing an increasing demand for highquality libraries. Significant effort has been invested at theWTCHG in improving the library preparation workflows through theintroduction of new equipment and automation to remove theidentified process bottlenecks. Adequate quality control of each ofthe many different type of libraries produced in our lab is crucial tothe success of the projects that we support. In order to maximizethe data output and quality, cluster generation on Illuminaplatforms requires accurate quantification. Alternative kits for


library quantification were evaluated on a Mx3005 qPCRinstrument. The performance of the QPCR NGS Library Quant Kitwas compared to that of other SYBR® Green based kits. Since wehave introduced the Agilent qPCR instrument and reagents in ourworkflow, we observed that approximately 85% of the librarysequenced at the WTCHG produced cluster densities within 20%from the expected value. Consumables for sequencing or unusuallibrary types have been often the cause for suboptimal clustergeneration. All the changes and improvements introduced in ourlab have contributed to lowering the costs and accelerate theturnaround time for projects due to reduced failure rates, however,the rapid and continuous changes in technology will requireconstant adjustments to fully utilize it.

Application of the Next Generation SequencingTechnologies at JCVI

Yu-Hui Rogers

J. Craig Venter Institute, United States of America

I will give an overview of the JTC research areas and how the nextgeneration sequencing technologies are being applied to supportthese activities. Specifically, I will discuss the sequencingtechnologies that are in use at JCVI including 454, Illumina andSOLiD, their capabilities and how we utilize different sequencingstrategies for different applications. I will also give an overview ofthe third generation sequencing technologies, including several ofthe single molecule sequencing technologies that are indevelopment and the potential impact of these technologies ongenomic research.

The use of qRT-PCR and high-throughputtranscriptomics for biomarker development

Michael W Pfaffl, Irmgard Riedmaier, Heinrich HD Meyer

Physiology Weihenstephan, TUM, 85354 Freising, Germany

The detection of growth promoting agents is a central topic inhuman doping surveillance and agricultural meat production. Inthe focus are the discovery and early verification of the illegal drugapplication, e.g. hormone cocktails or new xenobiotic growthpromoters. In routine screening, the residues of all known growthpromoters are detected by immuno based assays orchromatographical methods in combination with massspectrometry. To overcome the detection by these routine appliedscreening methods, in the previous years new xenobiotic drugsand application techniques were adapted. To enable a futureefficient tracing of misused anabolic substances it is necessary todevelop new screening and detection technologies for a broadrange of illegal hormones, including newly designed xenobioticanabolic agents. To achieve this, many innovative technologieshave been described for biomarker discovery and validation. Thistalk provides an overview of the latest transcriptomics basedtechnologies in biomarker discovery after illegal hormoneapplication. These –omics technologies can provide essentialinformation on the mRNA expression of hormone regulated genesacross multiple tissues, organs, treatment stages, andexperimental trials. It highlights recent gene expression results onmRNA and microRNA level derived from quantitative RT-PCR andnext generation sequencing. On the basis of the expressionresults reliable candidate biomarker were selected. To verify ifthese gene expression changes could act as first valid biomarkersbio-statistical methods were applied. Principal ComponentsAnalysis and Hierarchical Clustering were successfully used todemonstrate the potential of the transcriptomic approach for thedevelopment of a new screening system and to introduce newgene expression biomarkers.

Deep sequencing as diagnostic tool for highlypathogenic viruses

Aleksandar Radonic, Andreas Kurth, Wojtek Dabbrowski,Andreas Nitsche

Robert Koch Institute, Germany

With the advent of the real-time PCR nucleic acid-baseddiagnostics has become the gold standard for the identification ofviral and bacterial pathogens in clinical as well as in environmentalsamples. Real-time PCR is fast, reliable, and due to theapplication of specific “probes” it offers an additional level ofspecificity. Because of this pronounced specificity, however, PCR-based techniques may often fail to detect new or emerging

pathogens with differing or so far unknown genetic information.While multiplex PCR systems are a convenient approach tospecifically detect a wider variety of pathogens in one reactionvessel, generic PCR-based amplification systems offer a moreopen view and can even facilitate the detection of new pathogens.

Compared to electron microscopy, which provides an actualdiagnostic open view with serious restrictions regarding thedetection limit, recently metagenomic approaches based onmassively parallel sequencing techniques have promised to be amore sensitive valuable tool for the detection of unknownpathogens. Since it is technically possible to gain sequenceinformation of all pathogens present in a particular sample, themost challenging task is to identify the sequences of interest in thebulk of sequence data obtained by only one sequencing run. Inthis presentation the benefits and drawbacks of next generationsequencing as diagnostic tool will be discussed in comparison toconventional methods of virus detection.

High Resolution Transcription Profiling using Next-Generation-Sequencing identifies Diagnostic Markersfor Malignancy

Ralf Horres1,3, Björn Rotter1, Peter Winter1, MichalskiChristoph2, Friess Helmut2, Jüngling Ruth1,3, Günter Kahl3,Nicolas Krezdorn1, Klaus Hoffmeier1, Jörg Kleeff2

1GenXPro GmbH, Germany; 2Chirurgische Klinik und Poliklinik,Klinikum rechts der Isar, TU München; 3Goethe UniversitätFrankfurt, Institut für Molekulare Biowissenschaften

Since up to 95% of all transcripts are expressed at low levels,these rare transcripts are extremely important for a cell‘s fate andfunction. A SuperTag Digital Gene Expression (ST-DGE) Profilingapproach was established and applied for the analysis ofmalignant potential of pancreatic cancers. In contrast tomicroarrays ST-DGE is more reproducible, detects higher dynamicranges of gene expression, discovers natural antisense transcriptsand also counts rare transcripts, which are obscured in amicroarray’s background signal. The highly reliable geneexpression signatures allow choosing a subset of diagnostictranscripts converting the assay to a more clinically friendly PCR-based assay. This study aimed at analyzing processes underlyingthe tendency towards malignancy from chronic pancreatitis (CP) tobenign intraductal papillary mucinous tumor (IPMT) to highlymalignant ductal adenocarcinoma of the pancreas (PDAC). ST-DGE generated a complete survey of poly-adenylated transcriptsin excised normal, CP, IPMTs and PDAC pancreatic tissues. Boththe sense (s) and antisense (as) transcriptomes of CP and IPMTwere significantly different from normal pancreas, and all threedeviated from the transcript patterns characteristic for thetendency to malignancy, finally manifested in PDACs. A subset ofthese transcripts is now tested as prognostic and diagnostic qRT-PCR-based markers for malignancies in pancreatic tissue.GenXPro, est. 2005, is a service provider for the analysis ofnucleotide-based information content of any biological sample.The service portfolio includes the best available, focused andsensitive techniques, mostly using next generation sequencing,delivering qualitative and quantitatively most accurate results, atlowest possible costs. GenXPro offers full-service, “from tissue todata” including most suited bioinformatics approaches for theindividual next generation data handling. Our data can beconveniently analyzed using both our own “analysis suite” and /orexcel. Our analyzes include quality assessment, quantification,transcriptome mapping, Genome mapping, Gene Ontologyanalysis, plots , etc. to ease the analysis and publications. Ourtechnical service portfolio is headed by our patented „SuperSAGE“and „SuperTag Digital Gene Expression“ (ST-DGE) Profiling, forultra deep transcriptomics studies, but also includes RNAseq,normalization & sequencing of cDNA, microRNA analysis,epigenetics, copy number variation detection and genotyping aswell as qPCR service. GenXPro is involved in the development ofseveral diagnostic genetic markers for human diseases and inplant and animal breeding.


RNAi - microRNA - siRNA Applications

Time: Tuesday, 29/03/2011: 8:30am - 12:00pmLocation: Lecture hall 15Session Chair: Jo Vandesompele,

Jan Hellemans

Development of an ultra-high throughput long non-coding RNA qPCR screening system

Jan Hellemans1, Pieter Mestdagh2, Steve Lefever2, BarbaraD'haene1, Filip Pattyn2, Frank Speleman2, Jo Vandesompele1

1Biogazelle, Ghent, Belgium; 2Ghent University, Ghent, Belgium

Long non-coding RNAs (lncRNA) are an underexplored class ofnon-coding RNAs and have been shown to be implicated in healthand disease. They constitute a new class of biomarkers withdisease associated lncRNA signatures yet to be discovered. Inaddition, they open up a new approach to understand the functionand organization of the genome. The lack of a high-throughputplatform to detect and quantify lncRNAs has hampered their studyso far. To address this, we developed a new platform for ultra-highthroughput RT-qPCR analysis of long non-coding RNAs. In thepilot phase, we designed qPCR assays for +1000 lncRNAs basedon public sequence databases and lincRNA chromatin signatures.All assays were designed using state-of-the-art in silico qualitycontrols, followed by extensive empirical validation according toMIQE guidelines. Validation of this first set in relevant biologicalmodel systems demonstrated excellent sensitivity and specificityof the technology. Data processing was done using the qbasePLUS

software with an improved global mean normalization procedure tobetter remove technical variation. In conclusion, we havesuccessfully completed the first stage of the development processof an ultra-high throughput and low-volume RT-qPCR platform forthe quantitative detection of lncRNAs. The tool offers a uniqueway to investigate the expression patterns of lncRNAs in healthand disease.

Towards blood-based cancer screening using LNA™-enhanced microRNA qPCR

Ditte Andreasen, Niels Tolstrup, Jacob Ulrik Fogh, SørenJensby Nielsen, Kim Bundvig Barken, Adam Baker, PeterMouritzen

Exiqon, Denmark

microRNAs (miRNAs) constitute a recently discovered class ofsmall RNAs (typically 21-23 nt) that function as post-transcriptionalregulators of gene expression. Current estimates indicate thatmore than one third of the cellular transcriptome is regulated bymiRNAs, and miRNAs have been proposed to be masterregulators of cellular state. Indeed, changes in miRNA expressionpatterns have been associated with disease states, including adiverse array of human cancers. Furthermore, the high stability ofmiRNA in common clinical source materials (e.g. FFPE blocks,plasma, serum, urine, saliva, etc.) and the ability of miRNAexpression profiles to accurately classify disease states havepositioned miRNA quantification as a promising new tool for awide range of diagnostic applications. To facilitate discovery andclinical transfer of miRNA-based diagnostic markers, wedeveloped the miRNome-wide LNA™-based miRCURY LNA™Universal RT microRNA PCR platform with unparalleled sensitivityand robustness. Using a specialized design algorithm (availableas a web-tool for custom design of RNA species < 30 nt), we havedesigned assays for human and rodent miRNAs. The platformuses only 40 ng total RNA in just a single RT reaction to profile>700 human miRNAs in two predefined 384 well plates and thusallows high-throughput profiling of miRNAs from important clinicalsources without the need for pre-amplification. Followingscreening, it is possible to choose a sub-set of miRNA assays forvalidation to be formatted in 96- or 384-well plates using ourPick&Mix platform. The high sensitivity of the assays makes itpossible to do high quality microRNA expression profiling insamples that contain very little total RNA, such as sections offormalin fixed paraffin-embedded samples (FFPE) and bloodserum and plasma. Results will be presented demonstrating theapplication of the new qPCR method in the profiling of miRNAsfrom plasma as part of our efforts to develop molecular diagnostic

tests for treatment selection, diagnosis and monitoring of cancer.We will also show results demonstrating how selection of a properset of miRNA biomarkers in a Pick&Mix plate format can giveprecise classification of disease progression.

MicroRNA Target Validation with MISSION® 3'UTRLenti GoClone™ and Human MicroRNA Mimics

Nikos Hontzeas

Sigma Aldrich, United States of America

MicroRNAs (miRNAs) may regulate several hundreds of genes tocontrol a cell’s response to developmental and environmentalsignals. The validation of potential target genes is essential indetermining a miRNA’s role and function in these pathways. Herewe use MISSION® 3'UTR Lenti GoClone™ and Human MicroRNAMimics to demonstrate validation of known and conserved miRNAtargets. Known targets of hsa-miR-29b (MCL1), hsa-miR-124(MAPK14), and hsa-miR-373 (LATS2), and targets of conservedmiRNAs hsa-miR-373 (RBL2) and hsa-miR-10a, (HOXD10), weredown-regulated by their respective mimic. These resultdemonstrate that the use of MISSION® 3'UTR Lenti GoClone™

with Human MicroRNA Mimics is a viable option for miRNA targetvalidation.

Impact of different normalization strategies on miRNAprofiling experiments

Swanhild Meyer1, Sebastian Kaiser2, Carola Wagner3,Christian Thirion4, Michael Pfaffl1

1Physiology Weihenstephan, Technische Universität München,Germany; 2Department of Statistics, Ludwig-Maximilians-Universität München, Germany; 3IMGM Laboratories GmbH,Martinsried, Germany; 4Friedrich-Baur-Institute and Department ofNeurology, Ludwig-Maximilians-Universität München, Germany

Selection of the normalization strategy has significant impact onthe detection of differentially expressed microRNAs (miRNAs)from profiling experiments. Normalization techniques currently inuse for miRNA profiling analyses are in analogy to mRNA dataprocessing or are specifically modified and developed for miRNAdata. Studies evaluating the impact of normalization on miRNAprofiling experiments have focused on different profiling platformsor the comparison of normalization techniques within one platform.Here, we investigated the impact of seven different normalizationmethods (reference gene index, global geometric mean, quantile,invariant selection, loess, loessM, and generalized procrustesanalysis) on intra- and inter-platform performance of a one-colourhybridization-based platform (AGL array) and a multiplex RT-qPCR platform (TLDA) and validated results by singleplex RT-qPCR assays.

RNAi and gene expression profiling as a tool in cancerresearch

Michelle Plusquin1, An-Sofie Stevens1, Katrien De Mulder2,Frank Vanbelleghem1, Peter Ladurner2, Ann Cuypers1, TomArtois1, Karen Smeets1

1Hasselt University, Belgium; 2University of Innsbruck

As innovative model organisms in stem cell research, the flatwormspecies Schmidtea mediterranea and Macrostomum lignano wereused to characterize carcinogenic events. To discover potentialbiomarkers, RNAi knockdown and qPCR profiling were combined.Both cancer inducers as well as blockers were used anddifferential expression was compared. Underlying mechanismsinvolve oxidative stress related processes resulting in altered geneexpression profiles in function of (stem) cell proliferation. As heatshock proteins (HSP) appear to play a significant role herein, theeffect of a chemotherapeutic HSP blocker was evaluated. Thevalue of both RNAi experiments as well as gene expressionprofiling is highlighted in this presentation, and an overview ofcarcinogenic stress mechanisms and their interactions is given.


Absolute and relative quantification of placentalspecific microRNAs in maternal circulation in placentalinsufficiency related complications

Ilona Hromadnikova1, Katerina Kotlabova1, Jindrich Doucha2,Klara Dlouha3

1Third Faculty of Medicine, Charles University Prague, CzechRepublic; 2Clinic of Obstetrics and Gynecology, University HospitalMotol, Prague, Czech Republic; 3Institute for the Care of theMother and Child, Prague, Czech Republic

The primary goal of our study has been to identify placentalspecific microRNAs present in maternal plasma differentiatingbetween normal pregnancies and non-pregnant individuals. Theselection of appropriate pregnancy associated microRNAs with thediagnostical potential was based on following criteria: (1) detectionrate of 100 % in full-term placentas, (2) detection rate of above 67% in maternal plasma throughout gestation (at least 4 positivewells out of 6 tested wells) and (3) detection rate of 0 % in wholeperipheral blood and plasma samples of non-pregnant individuals.Initially, we tested microRNAs (miR-34c, miR-372, miR-135b andmiR-518b) which had been previously identified as pregnancy-associated miRNAs. Additionally we selected 16 other highlyspecific placental miRNAs (miR-512-5p, miR-515-5p, miR-224,miR-516-5p, miR-517*, miR-136, miR-518f*, miR-519a, miR-519d,miR-519e, miR-520a*, miR-520h, miR-524-5p, miR-525, miR-526aand miR-526b) from the miRNAMap database. Seven microRNAs(miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525and miR-526a) were newly identified as pregnancy associatedwith diagnostic potential. Further, we examined if extracellularplacental specific microRNAs (miR-520a*, miR-520h, miR-525 andmiR-526a) can differentiate pregnancies with placentalinsufficiency related complications from normal ones. Absoluteand relative quantification of placental specific microRNAs (miR-520a*, miR-520h, miR-525 and miR-526a) was determined in 50normal pregnancies, 32 complicated pregnancies (21preeclampsia with or without intrauterine growth retardation and11 IUGR) and 3 pregnancies at various gestational stages wholater developed preeclampsia w/o IUGR and/or IUGR using real-time PCR and the comparative Ct method relative to ubiquitousmiR-16. Both quantification approaches revealed significantincrease of extracellular placental specific microRNAs levels overtime in normally progressing pregnancies, however did notdifferentiate between normal and complicated pregnancies at thetime of preeclampsia and/or IUGR onset. Nevertheless, significantelevation of extracellular microRNAs was observed during earlygestation (within 12th to 16th weeks) in pregnancies with later onsetof preeclampsia and/or IUGR. To avoid instability of extracellularmicroRNAs in maternal plasma, prenatal monitoring should beperformed using only the samples with the storage life below twomonths. Early gestation extracellular microRNAs screening maydifferentiate between normal pregnancies and those who will laterdevelop placental insufficiency related complications.

Acknowledgements: This work was supported by grant projects MSM0021620806 and GAUK 260707/SVV/2010.

qbasePLUS: data analysis lunch seminar

Time: Tuesday, 29/03/2011: 1:00pm - 2:00pmLocation: Lecture hall 15Session Chair: Barbara D'haene

Jan Hellemans

qbasePLUS 2.0: the next generation of real-time PCRdata-analysis software

Barbara D'haene, Jo Vandesompele, Jan Hellemans

Biogazelle, Belgium

Biogazelle invites you to a 45 minutes live demonstration duringwhich we will showcase qbasePLUS 2.0, a major upgrade of themost powerful, flexible, and user-friendly real-time PCR data-analysis software.

First, we will walk you through the analysis of a small qPCRexperiment highlighting the need for quality control while handlingqPCR data. Tips and tricks provided during this demonstration will

guide you to highest quality results! Next, we will demonstratesome new 2.0 features, including sample grouping, new coremodules (e.g. copy number variant analysis), and alternativenormalization methods (e.g. global mean normalization for miRNAprofiling). Icing on the cake will be an introduction to the integratedstatistical analysis wizard which eliminates the need for in-depthknowledge about bio-statistics. This workshop will make you asavvy qPCR analyst in less than one hour, speeding up all yourfuture real-time PCR experiments!

Don’t miss out – a one-year license will be handed out to one ofthe attendees!

http://www.biogazelle.com

New qPCR Applications and Method Optimisation

Time: Tuesday, 29/03/2011: 2:00pm - 6:00pmLocation: Lecture hall 15Session Chair: Heinrich HD Meyer, Robert Loewe

Quantitative Immuno-PCR based on Imperacer:Technology, Platform & Analytical Applications

Jan Detmers, Mark Spengler, Michael Adler

Chimera Biotec GmbH, Germany

While conventional ligand-binding assay formats like ELISA orECL provide sufficient sensitivity in biological matrices to supportmany bio-analytical projects, a number of ligand-binding assayrequires ultra-sensitivity. Immuno-PCR (IPCR) combines the highspecificity of the antibody-antigen interaction with the exponentialsignal amplification by PCR as the most sensitive detectionmethod in molecular biology, to an ultra-sensitive ligand-bindingassay format. Due to exponential signal generation, assaysensitivity and broad detection range are the main advantages ofIPCR over conventional ligand-binding assays. Here, we present arange of case studies, making use of Imperacer®, an IPCR basedimmunoassay system, in combination with tailored sample dilutionprocedures to enhance assay robustness and precision byminimizing interfering effects from the sample matrix, endogenouscounterparts of the analyte or the biotherapeutic drug candidate inimmunogenicity assessment. This approach has successfullybeen demonstrated in fields like PK/PD, Biomarkers, ReplacementTherapy, Monitoring of Mucosal Vaccination, Virus Load Detectionand Anti-Drug Antibody Testing.

Quantitative Assessment of Chromatin Structure:Analysis of Epigenetically Regulated Gene Promoters.

Francisco Bizouarn

Bio-Rad Laboratories Inc, United States of America

Epigenetic processes, such as DNA methylation and histonemodification, control gene expression by altering chromatinstructure. Genes that are actively transcribed are associated with“open” or “accessible” chromatin regions; genes that aretranscriptionally silent are often in “closed” or “inaccessible”chromatin. We present a new assay, termed the EpiQ chromatinassay, that quantitatively assesses chromatin structure in culturedcells. The assay is novel, easy to perform, quantitative andproduces results on the day of cell harvest. We validated thisassay by analyzing 15 genes in four human cancer cell lines (4housekeeping genes and 11 genes that are epigeneticallyregulated in human cancer). We observe an excellent correlationbetween the level of gene expression and the chromatin structureof the corresponding promoter. Importantly, our data indicates thatgene promoters exist not just in “open” and “closed” chromatinstates, but also exist in intermediate states of chromatinaccessibility which act to fine-tune the level of gene expression.The EpiQ chromatin assay compliments and extends existingepigenetic assays and allows researchers to gain novel insightsinto mechanisms of gene regulation.


Quantification of specific mutation with HRM forregions of interest

Robert Loewe

GeneWake GmbH, Germany

HRM has been implemented on numerous instruments with manychemistry options. Despite this fact optimization and quantificationof e.g. mutational status has not yet reached most of the labs.Therefor a comparison of instruments and different chemistry wasdone. As a model system the SNP responsable for lactoseintolerance (rs4988235) was used to find optimal amplicon length(tested from 60 - 653bp). Also we tested the possibility to calculatethe mutational percentage for diagnostic purposes. The outcomepresented a potential solution for quantitative HRM with thepossible usage in screening minute sample material for mutationalstatus.

Quantitation of Rare Undifferentiated hESCs byMethylation-Specific CastPCR (MeS castPCR)

Jason Gioia, David Deng, Jeff Rosner, Caifu Chen


Human embryonic stem cells (hESCs) retain the remarkable abilityto differentiate into multiple different tissues and to self-renew invitro. These remarkable abilities make hESCs very attractive toboth scientific and clinical communities because they arepotentially a renewable source of a wide variety of human tissueswhich can be used for regenerative medicine, drug discovery andtoxicity testing. However, during the cell type-specificdifferentiation of hESCs, a small fraction of undifferentiated hESCsfrequently remain mixed with the differentiated cells. Theseundifferentiated hESCs pose serious risk by forming tumors aftertransplantation into patients. Therefore, it is important to detect &quantify rare, undifferentiated hESCs in therapeutic stem cellproducts. Based on distinct methylation patterns between hESCsand their differentiated cells in a set of genes, here we propose anew technology called methylation-specific castPCR (MeScastPCR) for accurate and sensitive quantification ofundifferentiated hESCs. This new technology will bridge the gap inhESC product QC and then overcome this major obstacle in stemcell therapy. CastPCR combines allele-specific TaqMan qPCRwith allele-specific minor grove binder (MGB) as blockers tosuppress effectively non-specific amplification from the wild typeallele. We have successfully designed and validated castPCRassays for ~100 SNPs or InDels. Results demonstrate thatcastPCR not only maintains wide dynamic range, high sensitivity,and reproducibility of TaqMan assays but also is able to detectone mutant in 100,000,000 wild-type molecules. Recently, Laurentet al. (2010) and Lister et al. (2009) presented a whole-genomecomparative view of DNA methylation using bisulfite sequencing ofhESCs and various differentiated cells. Based on their findings, wehave identified genes and genomic DNA regions such as HOXB6and others which show distinct DNA methylation patterns betweenhESCs and differentiated cells. These regions could be used asmethylation markers to distinguish between hESCs and theirdifferentiated (somatic) cells. It is important to note that DNAregions with 0% methylated in hESC and 100% in differentiatedcells are mostly desirable due to incomplete bisulfate conversion.MeS castPCR is aimed to detect converted CpG sites which areused to estimate % of hESC contamination in therapeutic cellproducts. Our results suggest that MeS castPCR is capable ofdetecting hESC contamination as little as 0.01% in differentiatedstem cell therapy product during clinical trials.

PrimeTime® Pre-designed qPCR Assays using ZEN™Internal Dark Quencher Chemistry

Scott D. Rose

Integrated DNA Technologies, United States of America

Well-designed qPCR assays require the careful consideration ofprimer placement, specificity, avoidance of single nucleotidepolymorphisms, oligonucleotide interactions and accurate meltingtemperature calculations. Using pre-designed assays shifts theburden of assay development from the researcher to themanufacturer. Unfortunately, some of the pre-designed assaysets commercially available today were built using outdateddatabases and primer design was done with older, less accurateTm algorithms. Even worse, some suppliers do not providesequence information to the end user that would enable the

researcher to evaluate the reagents in the context of their specificrequirements or for use in publication. IDT now offers fullyguaranteed PrimeTime® Pre-designed qPCR assays (for all genesin the human, mouse, and rat genome) that avoid cross reactivitywithin that genome, known single nucleotide polymorphisms,primer interactions, and gives full disclosure of all sequenceinformation as recommended by MIQE guidelines. By includingZEN double-quenched probes, these assays offer sensitivity to 10copies or lower with the added security of knowing precise probelocation with full sequence disclosure.

PrimerXL: high-throughput assay design for qPCR andamplicon based NGS

Filip Pattyn, Steve Lefever, Frank Speleman, JoVandesompele

Ghent University, Belgium

Primers are the cornerstone of many modern-age high-throughputnucleic acid analysis techniques ranging from real-timequantitative PCR (qPCR) to amplicon generation for next-generation sequencing. Various tools are currently available forautomated primer design, but most lack a thorough downstreamevaluation. A proper assessment of the multiple aspects affectinga primer assay's efficiency is a laborious and repetitive workinvolving manual interactions with different software applications.We automated the primer design process from top to bottom into aprocessing pipeline combining experimentally optimized designguidelines with a multi-faceted primer evaluation. This evaluationincludes checking for the presence of SNPs in the primerannealing sites to avoid sample dependent amplificationefficiencies, evaluating secondary structures in the primerannealing sites hampering efficient primer annealing and testingthe primer specificity which results in primers having an goodoverall efficiency. The pipeline is customizable, making it possibleto design primers fitting specific needs. In addition to thedevelopment of standard qPCR-primers, the pipeline allows todesign primers targeting a specific transcript variant or a subset oftranscript variants of a single gene or specific gene loci likeputative transcription factor or miRNA binding regions.Another field in which PrimerXL exceeds concurrent tools is thedesign of primers used in the preprocessing steps of ampliconbased next-generation sequencing. Until now, the application ofhigh-throughput amplicon sequencing has been hindered by thelack of tools capable in developing efficient tiling primers. To fillthis void, we have expanded our primer design pipeline with amodule to design tiling primers targeting all exonic sequences of aspecific gene. The generated primers produce more equimolaramplicon sets leading to a cost-efficient and sensitive variationdetection without extra sequencing efforts. The low variation inamplicon molarity outperforms the widely used array capturingtechnology employed to amplify specific target regions.

Hot Start dNTPs – Novel Chemistries for Use inAdvanced PCR Applications

Natasha Paul

TriLink BioTechnologies, Inc., United States of America

PCR is a widely used scientific tool whose specificity can beincreased by the use of Hot Start technologies. Although manyHot Start technologies exist, recently developed CleanAmp™dNTPs are a distinct approach that employs modified nucleosidetriphosphates with a thermolabile protecting group at the 3'-hydroxyl. The presence of the protecting group blocks lowtemperature primer extension, which can often be a significantproblem in PCR. At higher temperatures, the protecting group isreleased to allow for incorporation by the DNA polymerase andmore specific amplification of the intended target. These modifieddNTPs provide comparable performance to other Hot Starttechnologies and can be used with thermostable DNApolymerases to turn a reaction into a Hot Start version. Thisthermolabile chemistry can be applied to dNTP analogs such asdUTP, which is used in UNG decontamination methods, and 7-deaza-dGTP, which is used to amplify difficult GC-rich targets. Inaddition, further studies have led to the development of 3'-protecting groups that deprotect more quickly than the current 3'-modification group, allowing these modified dNTPs to be used infast PCR. With the evolving chemistry of the hot start dNTPs, theareas of application benefiting from the versatility and flexibility ofthis technology continue to grow.


PCR-Luminex Based Detection of 28 Enteropathogens

Mami Taniuchi1, Jie Liu1, Shihab Sobuz2, William Petri1,Rashidul Haque2, Eric Houpt1

1University of Virginia, United States of America; 2InternationalCentre for Diarrhoeal Diseases and Research, Bangladesh

Background: Given the range of potential enteropathogensimplicated in diarrhea, we developed a PCR-Luminex based assayto detect 28 most common organisms associated with diarrhea.The enteropathogens included were Cryptosporidium spp.,Entameoba histolytica, Giardia, Microsporidia, Cystoisospora,Cyclospora, Ascaris, Hookworms, Strongyloides, Tricuris, EAEC,EHEC, EIEC, EPEC, ETEC, Astrovirus, Norovirus G1 and G2,Rotavirus, Sapovirus, Adenovirus, and an internal control. Thispanel was used in a prospective study to determine etiology ofdiarrhea in Bangladeshi children in the first year of life. Methods:Specific primers and probes were designed for the organismsusing targets which are conserved. DNA or RNA purified fromstool was amplified using biotinylated primers, followed byhybridization to amine-modified probes covalently linked tocarboxylated spectrally-distinct microspheres, followed by additionof streptavidin PE to detect specifically-bound amplicon. Luminexresults are reported as corrected mean fluorescence intensity(cMFI) normalized to background, where cMFI > 2.5 was utilizedas a “present” call with the exception of Cryptosporidium,Strongyloides, all EC, and all viruses where cMFI values were 7.3,9.0, 10.0, and 2.0, respectively. Results: Performance of theassays yielded 95% to 100% sensitivity and specificity versus theassays performed via real-time PCR. We then applied the tests ina prospective study of 147 children from Mirpur, Bangladeshfollowed monthly for the first year of life. 83% of the children hadat least 1 episode of diarrhea in the first year of life and 33% had 4or more. The number of pathogens that were detected increasedas the number of diarrheal episodes increased (e.g., from 1.7 to2.5 from first diarrheal episodes to 4th or later diarrheal episodes,P <0.05). Rotavirus, ETEC, EAEC, and were the leadingpathogens detected during the first diarrheal episodes, whileAdenovirus, Shigella, ETEC, EPEC, and Giardia emerged ascommon in the later episodes. Conclusion: This Luminex basedassay for the major enteropathogens offers sensitive and specificdetection similar to real time PCR. When applied to field studies inendemic areas, a singular etiology of diarrhea is difficult todetermine due to the frequency of mixed infections, and multiplepathogens may be the norm. Pathogens appear to accumulate inchildren that develop recurrent diarrhea.

Simple analytical and experimental procedure forselection of reference genes for qPCR normalizationdata

Rodrigo Manjarin1, Nathalie Trottier1, Patty Weber2, JamesLiesman1, Nathanael Taylor1, Juan Pedro Steibel1,3

1Department of Animal Science, Michigan State University, UnitedStates of America; 2Department of Large Animal Clinical Sciences,Michigan State University, United States of America; 3Departmentof Fisheries and Wildlife, Michigan State University, United Statesof America

Variation of cellular activity in a tissue induces changes in RNAand DNA concentration between samples, which may affect thevalidity of mRNA abundance of target genes obtained with real-time quantitative PCR (qPCR) analysis. A common way ofaccounting for such variation consists of the use of a referencegene as a normaliser. Programs such as geNorm can be used toselect suitable reference genes, although a large set of genes thatare not co-regulated must be analyzed to obtain accurate results.The objective of this study was to propose an alternative analyticalprotocol to assess the invariance of reference genes in porcinemammary tissue using mammary RNA and DNA concentrationsas correction factors. Mammary glands were biopsied from 4 sowson d 110 of gestation (pre-partum), on d 5 (early) and 17 (peak) oflactation, and on d 5 after weaning (post-weaning). Relativeexpression of seven potential reference genes, API5, MRPL39,VAPB, ACTB, GAPDH, RPS23 and MTG1, and one candidategene, SLC7A1, was quantified by qPCR using a relative standardcurve. The response in cycles to threshold at each stage oflactation was tested using a linear mixed model fitting RNA andDNA concentration as covariates. Results were compared to thoseobtained with geNorm analysis and genes selected by eachmethod were used to normalize SLC7A1. Quantified relativemRNA abundance of API5, GAPDH and MRPL39 remained

unchanged (P > 0.1) across stages after correcting with RNA andDNA concentration, whereas geNorm analysis selected MTG1,MRPL39 and VAPB as best reference genes. There were nodifferences between normalization of SLC7A1 with genes selectedby the proposed analysis protocol or by geNorm. In conclusion,the proposed analytical protocol and geNorm selected differentreference genes, but SLC7A1 fold changes did not differ whenusing genes obtained under either method. These results indicatethat among our set of candidate genes, there is more than onesuitable reference gene. The proposed method, however, has thestrength of allowing testing each potential reference geneindividually and sequentially, so analysis of remaining genes canbe spared as soon as a suitable reference gene is identified.

Wednesday 30th

March 2011

Pre-PCR: Pre-analytical Steps

Time: Wednesday, 30/03/2011: 9:00am - 12:00pmLocation: Lecture hall 14Session Chair: Jo Vandesompele, Karl H. Hasenstein

Measurable impact of RNA quality on gene expressionresults from quantitative PCR

Joëlle Vermeulen1, Katleen De Preter1, Steve Lefever1, JustineNuytens1, Fanny De Vloed1, Stefaan Derveaux1, JanHellemans2, Frank Speleman1, Jo Vandesompele2

1Ghent University, Ghent, Belgium; 2Biogazelle, Zwijnaarde,Belgium & Ghent University, Ghent, Belgium

Compromised RNA quality is suggested to lead to unreliableresults in gene expression studies. Therefore, assessment of RNAintegrity and purity is deemed essential prior to including samplesin the analytical pipeline. This may be of particular importancewhen diagnostic, prognostic or therapeutic conclusions depend onsuch analyses. In this study, the comparative value of six RNAquality parameters was determined using a large panel of 740primary tumour samples for which real-time quantitative PCR geneexpression results were available. The tested parameterscomprise microfluidic capillary electrophoresis based 18S/28SrRNA ratio and RNA Quality Index value, HPRT1 5’-3’ differencein quantification cycle (Cq) and HPRT1 3’ Cq value based on a5’/3’ ratio mRNA integrity assay, the Cq value of expressed Alurepeat sequences, and a normalisation factor based on the meanexpression level of 4 reference genes. Upon establishment of aninnovative analytical framework to assess impact of RNA quality,we observed a measurable impact of RNA quality on the variationof the reference genes, on the significance of differentialexpression of prognostic marker genes between two cancerpatient risk groups, and on risk classification performance using amultigene signature. This study forms the basis for further rationalassessment of RT-qPCR based results in relation to RNA quality.

Isolation and Analysis of Circulating Nucleic Acids:Technical Advances

Martin Horlitz

QIAGEN GmbH, Germany

Circulating DNA fragments and specific messenger RNAsoriginating from malignant tumors, a developing fetus, but alsoviral or bacterial infections are present in the cell-free nucleic acidsin plasma, serum and other body fluids such as urine. Access tothese nucleic acids for analysis could allow for specific detectionof certain disease states based on a simple blood sample.Circulating cell-free nucleic acids show distinctive properties: Theyare present in plasma and serum mostly as shorter fragments ofless than 500 bp or nt in size. The concentration of suchcirculating, cell-free DNA and RNA in a plasma or serum sample islow (≈1–100 ng/ml) compared to cellular materials and variesconsiderably between different individuals. Because of theirfragmented nature and low concentration, circulating nucleic acidspresent a particular challenge for efficient extraction/purification


and quantification by qPCR. We present data on solutions for thefollowing critical problems concerning the analysis of circulatingnucleic acids for research and molecular diagnostic applications:

- Pre-analytical workflow (blood processing) for analyzingcirculating nucleic acids

- Optimization of ccfDNA extraction from plasma samples: lowtarget concentrations require the efficient ccfDNA enrichment fromlarger sample volumes (1-5 ml)

- Use of an internal control to monitor the ccfNA extractionperformance

- Avoiding qPCR Quantification bias when working withfragmented target nucleic acids

- Assessment of ccfDNA fragment size distribution in plasmasamples by comparing qPCR results based on target amplicons ofdifferent sizes

Furthermore, we will present data on the extraction and qPCR-based quantification of circulating fetal DNA and mRNA frommaternal plasma samples obtained at the end of the first trimesterof pregnancy.

Solid Phase Gene Extraction – Sampling of mRNAfrom living systems

Karl H. Hasenstein, Min Chen

Univ. Louisiana Lafayette, United States of America

Understanding gene expression and regulation especially on acellular level requires rapid, localized and ideally repeatedsampling of mRNA. Current technologies inevitable rely on theextraction of tissue, cytoplasm aspiration, or cell lysates, all ofwhich pose problems because of unspecific sampling,contamination, and lack of repeatability. These methods aredestructive and do not distinguish between genomic DNA andRNA. Moreover, extracted mRNA is typically contaminated byextracted cytoplasm, nuclear DNA, or other compounds, and therequired purification leads to loss of especially low-abundantmRNA. Biological systems, where all regulatory and responsesteps take place in a single cell are thus not accessible tolocalized gene expression studies. This predicament lead to thedevelopment of a needle-based mRNA extraction system thatprovides high temporal and spatial resolution that allows forrepeated removal of mRNA from living material. This development(dubbed Solid Phase Gene Extraction, SPGE) can be designed tohybridize with gene-specific or generic (oligo-dT) sequences. It isnot species-specific and can be employed to access tissue orsingle cells. We demonstrate the versatility and validity of thisnovel RNA extraction technique by simultaneously profiling nanosand bicoid mRNA in individual Drosophila eggs. The distribution ofthese genes resulted in the same longitudinal distribution aspreviously described distribution profiles. The low impact of SPGEsampling was underscored by the normal development ofrepeatedly sampled eggs. Actin isoforms in germinating seedlingsvaried in the apical tissue of roots as a function of imbibition andpermits discrimination between newly synthesized and maternalgenes. Gene expression in human tumor tissue suggests thatSPGE has the potential to supplement or eliminate biopsies.Examination of several genes that are typically used as referencesin gene expression studies show that SPGE is more sensitive thantissue-based extraction and has the potential to function as a self-referencing system. These data demonstrate the universality ofSPGE as a simple, generic, analytical, and diagnostic procedure.

Impact of basepair mismatch in primer annealing onqPCR assay performance

Steve Lefever, Pattyn Filip, Speleman Frank, Hellemans Jan,Vandesompele Jo

Center for Medical Genetics Ghent, Ghent University, Belgium

With the onset of the 1000 Genomes Project that aims to explorehuman genetic variation, a multitude of new single nucleotide(SNP) polymorphisms have emerged. Therefore, the density anddistribution of known variants has increased significantly andhampers design of primers in regions devoid of SNPs.

Importantly, perfect primer/target complementarity is believed tobe crucial in all samples since the presence of a SNP in the primerannealing region may affect the result of a quantitative polymerasechain reaction (qPCR). In the worst case, absence of amplificationof the variant allele has been reported in literature. However, theimpact of such a mismatch in relation to its location in the primerand local sequence context has not been systematically studied.

To address this important issue, we have designed a study tosystematically and empirically assess the relation between thepresence of a mismatch in the last five 3’ end bases of a PCRprimer and various qPCR parameters, such as efficiency and Cq-value. The study is based on a constant reverse primer and a setof nine walking forward primers, located up to four bases up- ordownstream from a starting position, where single base pairmismatches have been introduced at the 3’ end final base. 36forward primers and 16 synthetic oligonucleotide templates (goodfor 576 distinct and informative match/mismatch reactions) weredesigned to prevent secondary structure formation, self-annealingand primer dimerization. Different qPCR reaction mixes withvariable sensitivity towards primer mismatches are evaluated.Data analysis is currently being finalized and results will bepresented at the symposium.

Gene Expression profiles from FFPE samples withimproved RNA decrosslinking technology A casestudy: Molecular profiling of breast cancer fromformalin-fixed, archival material

Guido Krupp1, Susanne Quabius2, Rolf Jaggi3

1AmpTec GmbH, Hamburg, Germany; 2Institute for Immunology,University Klinikum UK-SH, Kiel, Germany; 3Dept. ClinicalResearch, University of Bern, Bern, Switzerland

We have developed a novel demodifaction/decrosslinking protocolfor RNA recovery from archival (FFPE) material. The resultingFFPE RNA quality is superior to RNA obtained with othercommercial FFPE RNA isolation kits: larger RNAs can berecovered, and RT-qPCR data demonstrate less variability andlower Cq values. This FFPE RNA is suitable for differential geneexpression measurement by qPCR, high concordance withparallel RNA samples from fresh-frozen tissues was observed.Prognosis of breast cancer is determined by clinicopathologicaland molecular factors. We developed and validated molecularscores reflecting the hormone status (ER, PGR, HER2 scores)and the proliferation status (PRO score) of breast cancer cells.The scores can be combined to an overall RISK score. Molecularscores are independent prognostic parameters, they werevalidated in postmenopausal patients with estrogen receptorpositive breast cancer. Multivariate analysis revealed that PROand RISK scores outperform conventional parameters (histologicalgrading and Ki-67 labeling index). Molecular scores are based onroutine pathological material, testing can be implemented easilyinto routine diagnosis.

DEPArray™: a novel platform to identify, isolate andcollect very rare cells

Pim van der Aar1, Manuela Banzi2

1Dync B.V., Netherlands, The; 2Silicon Biosystems SpA

The DEPArray A300K system, uses a microelectronic chipintegrating an array of 307,200 tiny electrodes, implementing up to76,800 moving dielectrophoretic-cages for software-controlled cellsorting and manipulation. The chip is embedded in a microfluidicdisposable cartridge enabling multiple recoveries of individual orgroup of cells, as well as manipulation of cells and/or microbeadsfor cell biology experiments.

Cells injected in the chip are trapped in an array of field cages.Images are then acquired, and cell selected for sorting ormanipulation with the following unique features:

- sort by morphological parameters such as shape, nucleus tocytoplasm ratio, fluorophores co-localization, thanks to imagebased selection

- no contamination from spurious events verifying imagesassociated to cells selected from the scatter plot or histogram,including up to 5 fluorescence channels and bright-field

- no need for a-priori thresholds, pick-up only best cells availablein the sample

- sort specific rare-cells, based on relative fluorescenceparameters combined with morphology

- sort cells from a suspension where the total cell count isextremely low (0-100,000)

DEPArray is compatible with PBS and culture media. This allowsone to manage live cells, in physiological conditions, without theneed for special low-conductivity buffers.


With respect to other cell manipulation techniques such as opticaltweezers, DEPArray™ has the key advantage that

1) cells need not to be in the field of view of the microscope to bemanipulated

2) a massive amount of cells can be trapped and managed step-by-step, deterministically, under software control

These advantages translate in a whole new world of possibilitiesbeyond sorting, such as for example

- to run experiment of cell-cell interaction with exquisite control ontiming and parallelism of the interactions, or

- to stimulate cell receptors with functionalized microbeads, in acontrolled way

New qPCR Applications in Molecular Diagnostics

Time: Wednesday, 30/03/2011: 13:30pm - 4:30pmLocation: Lecture hall 14Session Chair: Andreas Nitsche

Heinrich HD Meyer

Detection and classification of microorganisms bycombination of qPCR and pyrosequencing

Kati Schroeder, Andreas Nitsche

Robert Koch-Institut, Germany

Microbial and viral typing is usually performed by Sanger DNAsequencing. Pyrosequencing (PSQ) is a new sequencing-by-synthesis method allowing a fast and reliable detection ofpolymorphisms and sequence variations between differentspecies. Performed in combination with quantitative PCR, PSQcan verify positive qPCR results and concurrently identify thepathogen species immediately after the PCR run is finished. Tocombine qPCR with PSQ, target regions within the respectivepathogen genome were amplified in parallel by qPCR with onebiotinylated primer for each assay. Positive qPCR results indicatedthe presence of a pathogen. Additionally, biotinylated DNA strandsof PCR products were separated and purified on Streptavidin-coated beads and hybridized to a sequencing primer. SubsequentPSQ generated a pyrogram with a specific sequence that allowedclassification of the pathogen by local BLAST search. As differentqPCR and corresponding PSQ assays can be easily performed inparallel, a broad range of pathogens can be detected rapidlyallowing accurate and reliable microbial and viral diagnostics insamples of unknown infectious origin.

FRET primer for single nucleotide polymorphism (SNP)genotyping

Choy Len Fong1, Fiona Ng1, Kanaga Sabapathy2, Kim Halpin1

1Life Technologies, Singapore; 2Laboratory of MolecularCarcinogenesis, National Cancer Centre, Singapore

Single nucleotide polymorphisms (SNPs) have become one of themost important objects of medical research. They have thepotential to identify new drug targets, explain individual differencesin the effectiveness of drugs, and susceptibility to disease.However the identification of SNP profiles by high resolution melt(HRM) is currently limited. The goal of this study was to developan accurate HRM assay which employs the use of fluorescenceresonance energy transfer (FRET) primers for identifying SNPs. AFRET primer is internally labeled with a fluorescence dye at athymine (T) nucleotide. A Fret primer assumes a randomly coiledstructure as a single stranded primer, resulting in the reporter dyeand quencher being in close proximity, hence quenchingfluorescence. Upon Fret primer extension, a rigid duplex forms,resulting in reporter dye and quencher separation and henceincreased fluorescence which is ideal for real-time PCRquantification and melt curve study. In this study, we describe thefeasibility of using a Fret primer for genotyping SNPs with HRM-based genotyping, offering an alternative to an intercalating dye(i.e. SYTO-9)-based platform. A Fret primer targeting the p53codon 72 SNP site was designed and tested for the ability to

discriminate the two SNP homozygous populations by HRM on areal-time PCR platform. p53 is currently the most frequentlymutated tumour-suppressor gene in human cancers. A SNP incodon 72 of p53 results in either an arginine (Arg) or proline (Pro)residue, and has been demonstrated to affect p53 function. Ourresults show that the FRET primer coupled with HRM analysisreliably enables discrimination between Arg/Pro SNP in 18peripheral blood sample of cancer patients. The FRET primersexhibit comparable sensitivity and expansive dynamic range ascompared to SYTO-9 based HRM platform.

qPCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2 (AAV2) ITR-Sequences

Christine Aurnhammer, Maren Haase, Anja Ehrhard, MartinHäusel, Ingrid Huber, Nadine Muether, Hans Nitschko, UlrichBusch, Andreas Sing, Armin Baiker

Bavarian Health and Food Safety Authority (LGL), Germany

Background: Viral vectors based on adeno-associated virusserotype 2 (AAV2) constitute promising tools in human genetherapy. The inverted terminal repeats (ITRs) within the viralgenome are the only cis-acting viral elements required forfunctional AAV2 vector generation and constitute the lowestcommon denominator of all AAV2-based vectors. However, so far,no PCR-based method for the detection and quantification ofAAV2-ITRs could be established due to their extensive secondaryhairpin structure. Current PCR-based methods are thereforepredominantly targeting vector-encoded transgenes or regulatoryelements. Methods: We established an AAV2-ITR sequence-specific quantitative PCR (qPCR) method. Primers and BBQ-labeled probe are located within a particular subregion of the ITRsequence and have been designed to detect wild type AAV2 andAAV2-based vectors. Results: This method is suitable for theevidence of both, single-stranded (genomic) DNA derived fromAAV2 vector particles and double-stranded DNA derived fromproducer plasmids. The linear dynamic range could be shown for102 to 107 copies and the detection limit determined as 50 copies.A practical approach for the analysis of putative cross reactivitiesagainst closely related AAV serotypes utilizing synthetic oligonucleotides revealed some cross reactivity against orthologoussequences of AAV1, 3, 6 and 7 but not against AAV4, 5, 8 and 9.For AAV vector production adenovirus type 5 (Ad5) is often usedin terms of its helper virus properties. Therefore, we furtherinvestigated the specificity of our qPCR method using Ad5-DNAand could prove the method to result in no detectable crossreactivity with Ad5. Conclusion: This method comprises the firstqPCR system facilitating the detection and quantification of AAV2-ITR sequences. Since this method can be applied for all AAV2-based vectors in a “one for all”-based manner, it will significantlysimplify AAV2 vector genome titrations in the future.

Cost-efficient detection of Mycobacterium aviumsubsp. paratuberculosis in faeces by quantitative realtime PCR and development of a predictive model forfighting the disease

Petr Kralik1, Iva Slana1, Alena Kralova1, Vladimir Babak1,Robert H. Whitlock2, Ivo Pavlik1

1Veterinary Research Institute, Czech Republic; 2University ofPennsylvania, School of Veterinary Medicine, PA, USA

Mycobacterium avium subsp. paratuberculosis (MAP) causeslosses in ruminants especially in cattle. The conventional controlprogrammes are based on the traditional culture that suffers fromthe long time required for the incubation. In this study we haveaimed on two main issues. Firstly, we have developed andoptimised a reliable and cost-efficient DNA isolation procedurefrom faeces that could be coupled with previously developedIS900 and F57 quantitative real time PCR (qPCR) for the MAPdetection. To determine the quantity of MAP as precise aspossible, the recovery of MAP DNA from the spiked faecalsamples was established. It ranged from 29.1 to 102.4% of theinput amount of MAP with median 37.9%. The limit of detectionwas determined to be 1.03 × 104 for F57 qPCR and6.87 × 102MAP cells per gram of faeces for IS900 qPCR,respectively. The developed technique for DNA isolation wascoupled with IS900 qPCR and compared to traditional MAPculture using a cohort of 1906 faecal samples examined from 12dairy cattle farms in our laboratory. From those 1906 originalfaecal samples, 875 were positive by IS900 qPCR and 169 by


culture. None of the culture positive samples was negative byIS900 qPCR. This data facilitated development of a predictivemodel capable of estimating the probability of being culturepositive by estimating the absolute number of MAP per gram offaeces as determined IS900 qPCR without performing the culture.

This work was supported by the EC (ParaTBTools), the Ministry ofEducation, Youth and Sports of the Czech Republic “AdmireVet” (CZ1.05/2.1.00/01.0006 and No. ED 0006/01/01) and the Ministry of Agricultureof the Czech Republic (Grants Nos. MZe0002716202 and QH81065).

Development of a novel duplex real-time PCR assayfor plasma DNA quantification and its comparison toother methods

Shiyang Pan, Dan Chen, Peijun Huang, Bing Gu, Fang Wang,Jian Xu, Chun Zhao, Yongqian Shu, Di Yang

The first affiliated hospital with Nanjing medical university, China,Peoples Republic of

BACKGROUND: It has long been known that plasma DNA ispresent in healthy and diseased individuals and manyinvestigators have been attracted to this emerging field. However,a direct comparative analysis of the abundant plasma DNA dataavailable is often prevented by variability in sample storage,sample processing, and protocols for DNA extraction. Therefore, amore accurate and stable assay with lower uncertainty is neededin conjunction with a multi-center study. METHODS: In this study,a duplex real-time PCR assay with an internal control wasdeveloped as a novel method for quantification of plasma DNA.The detect limit, precision, accuracy and stabilitity of this novelassay was then evaluated and compared to other two popularplasma DNA quantification methods by real-time PCR withexternal standards and by direct fluorescent PicoGreen staining.Furthermore, the plasma DNA concentrations of 1,187 healthyChinese adults have been determined by the novel assay.RESULTS: The detect limits of novel duplex real-time PCR, real-time PCR with external standards and PicoGreen assay were 0.1ng/ml (R2 = 0.96, P < 0.0001), 0.1 ng/ml (R2 = 0.96, P < 0.0001)and 1.0 ng/ml (R2 = 0.91, P < 0.0001), respectively. For plasmawith concentration of 1.0 ng/ml, the CV values of duplex real-timePCR assay, real-time PCR with external standards and Picogreenassay were 53.0%, 60.3% and 96.7%, respectively. In therecovery test, there was a higher correlation of the plasma DNAconcentrations determined by the duplex real-time PCR assaywith the input DNA levels (R2 = 0.97, P < 0.0001) than those of thereal-time PCR with external standards (R2 = 0.85, P < 0.0001) andPicoGreen assay (R2 = 0.92, P < 0.0001). Although remarkabledifferences in plasma DNA concentrations determined by the real-time PCR with external standards and PicoGreen assay existed insamples purified by the three different protocols (P = 0.0041 and0.0000), there was no statistically significant difference in plasmaDNA concentrations among the methods by using our duplex real-time PCR assay (P = 0.5821). The median plasma DNAconcentration of females (16.9 ng/ml) was significantly lower thanmales (22.6 ng/ml; Mann-Whitney two-sample rank sum test, P <0.0001). Within the 95% confidence interval, the normal referenceinterval of the plasma DNA concentration was 0~50 ng/ml formales and 0~40 ng/ml for females. CONCLUSIONS: This newlydeveloped plasma DNA quantification method with the power ofeliminating variables introduced during plasma sample processingallows more sensitive, repetable and accurate quantitativemeasurement, and has a very promising future in clinicalapplication for diagnosis and disease monitoring.

Autocrine and paracrine role of leptin andthrombopietin in controlling ovarian function in cow

Mihir Sarkar1,2, S Schilffarth1, D Schams1, HHD Meyer1, M WPfaffl1, S Ulbrich1, B Berisha1,3

1Technical University Munich, Germany; 2Physiology &Climatology, Indian Veterinary Research Institute, Izatnagar,Bareilly, Uttar Pradesh, India; 3Faculty of Agriculture andVeterinary, University of Prishtina, Prishtine, Kosovo

Leptin, the hormonal product of the obese (ob) gene, circulates inthe blood at levels paralleling those of fat reserves and regulatessatiety. In cattle, leptin has also been implicated in the control ofovarian function, but its local production in the ovary and role inthe control of ovarian function in autocrine/paracrine manner isunknown. Similarly, Thrombopoietin (TPO) is known to be involvedin mega-karyocytopoiesis, but its role in the control of ovarianfunction is unknown in cattle. The aims of this study were to

demonstrate the expression of Leptin, its receptor (Ob-R), TPOand its receptor (c-MPL) in detail in bovine corpus luteum (CL)obtained from different stages of the oestrous cycle and duringpregnancy – and to demonstrate that leptin/Ob-R and TPO/ c-MPLsystems are expressed clearly in bovine follicles. Real-time RT-PCR (qPCR) and ELISA were applied to investigate mRNAexpression of examined factors and leptin & TPO protein,respectively. In general, we demonstrated leptin and its receptortranscripts and leptin protein are consistent with in vivoluteinisation of bovine CL and decline coincidental with lutealregression. The highest co-expression of leptin/Ob-R system wasobserved in TI and GC of the smallest follicles with E2concentration <0.5 ng/ml followed by significant down regulation ingrowing follicles with the increase of follicular size and E2 contentin the follicular fluid. Furthermore, expression of the leptin/Ob-Rsystem does not show any significant variation in the CLthroughout pregnancy. In this investigation, increases in theconcentrations of TPO protein and the mRNA expression of TPOand c-MPL were noticed during both early luteal stage and lateluteal stage of the oestrous cycle. Furthermore, the expression ofTPO/ c-MPL system does not show any significant regulation inthe CL throughout pregnancy. Highest co-expression of TPO/ c-MPL system in both theca interna (TI) and granulosa cells (GC) insmall follicles (<10 mm in diameter) was observed in this studythat may suggest the possible role of TPO/ c-MPL system inproliferation of TI and GC cells. To conclude, our results are thefirst to demonstrate the possible involvement of locally producedleptin/Ob-R and TPO/ c-MPL system in the bovine ovary,suggesting possible involvement of leptin/Ob-R system in thefunction and/or development of the CL and growth of small folliclesin an autocrine/ paracrine fashion and of TPO/ c-MPL system as a‘physiological filter’ in bovine ovary where they may promote cellselection by inducing proliferation of viable cells and scavengingnon-viable cells and thereby may play an important role inmodulation of ovarian function.

ULTRA-RAPID REAL-TIME PCR for the detection ofviral diseases in honeybee

ByoungSu Yoon, MiSun Yoo, JiNa No, Van Phu Nguyen

Kyonggi University, Korea, South (Republic of)

Viral diseases in honeybee are very difficult to be identified. Anovel micro PCR-based detection method, termed ultra-rapid real-time PCR (URRT-PCR), was applied to the development of arapid detection for pathogenic virus in honeybee quantitatively,such as IAPV, BQCV. In the URRT PCR assays showed highsensitivities and accuracies in the both standard assays andinfected bees. In the application of URRT PCR detection frominfected honeybee, the detection time was only within 8 min,including melting temperature analysis, performing 35 cycledPCR. This novel detection method is one of the most rapid real-time PCR-based diagnostic tools and is expected to be applied tothe development of a rapid detection for various pathogens.

Development of quantitative triplex real time PCR forthe simultaneous detection of Mycobacterium aviumsubsp. avium and M. a. subsp. hominissuis

Iva Slana, Maria Kaevska, Petr Kralik, Alice Horvathova, IvoPavlik

Veterinary Research Institute, Czech Republic

Mycobacterium avium subsp. avium (MAA) and M. a. subsp.hominissuis (MAH) belong to the Mycobacterium avium complexand are frequently associated with diseases in animals andhumans. In animals it causes tuberculous lesions inparenchymatous organs. Infections with MAA and MAH in humansare rather scarce, but when present they cause severecomplications that lead to the chronic stage or sometimes even tothe death of individual. The aim of this study was to develop asystem for rapid and accurate quantitative real time PCR (qPCR)identification and quantification of MAA and MAH. The developedtriplex qPCR reaction was based on the simultaneous detection ofspecific insertion sequences, IS901 and IS1245 and an internalamplification control. The specificity and sensitivity of the qPCRwere determined as well as the limit of detection for bothpathogens isolated from tissue samples. In order to quantify bothpathogens as precise as possible, the recovery of MAA and MAHcells after DNA isolation was established. To test the triplex qPCRassay coupled with the DNA isolation, tissue samples from 22 per


os artificially infected pigs, of which ten were infected with MAA,ten with MAH and two were present as a negative control groupwere tested. From each animal, 21 different tissue samples aswell as blood were tested by microscopy, culture and qPCR. Inboth groups of experimentally infected animals, the newlydeveloped triplex qPCR assay proved to be more specific andsensitive in comparison with the other methods used. Contrary toculture examination, triplex qPCR confirmed the infection in allanimals infected with MAA, and in eight animals infected withMAH. In conclusion, we developed a quick and sufficientlysensitive triplex qPCR for MAA and MAH detection in tissuesamples that represents a suitable alternative to the culture.

This work was supported by the Ministry of Education, Youth and Sports ofthe Czech Republic “AdmireVet” (CZ 1.05/2.1.00/01.0006 and No. ED0006/01/01), the Ministry of Agriculture of the Czech Republic (Grant No.MZe0002716202) and the EU grant PathogenCombat.

Rapid KRAS, EGFR, BRAF and PIK3CA MutationAnalysis of Fine Needle Aspirates using allele-specificqPCR

Ronald van Eijk, Jappe Licht, Hans Morreau, Tom van Wezel

Leiden University Medical Center, Netherlands, The

Fine needle aspiration based novel techniques for the diagnosisand staging of diverse cancers have been incorporated intocancer staging guidelines. The concordance rate of the mutationanalysis between these cytological aspirates and histologicalsamples obtained by surgical staging is often unknown. Therefore,we studied the extent to which allele-specific quantitative real-timePCR with hydrolysis probes could be reliably performed on fineneedle aspirates by comparing the results with histologicalmaterial from the same patient. Assays for the detection of sevendifferent KRAS, three PIK3CA and one BRAF variant togetherwith two different EGFR assays have been used on a series ofpatients for whom cytological and histological material wasavailable. Raw data from the LC480 or CFX384 software wereimported into an in-house–created Microsoft Excel 2003spreadsheet to define the mutation status. We demonstrated thatthese standard molecular techniques can be accurately applied onfine needle cytological aspirates.

Data Analysis: qPCR BioStatistics &BioInformatics

Time: Wednesday, 30/03/2011: 9:00am - 12:00pmLocation: Lecture hall 15Session Chair: Michael W Pfaffl, Ales Tichopad

Interpretation requires context - making sense out ofgene lists and networks.

Philip Zimmermann

ETH Zürich, Switzerland

Many functional genomics and systems biology approaches resultin lists of genes with common properties. The interpretation of alist of genes is crucial in understanding the results of such anexperiment. Typical approaches are to study gene enrichment ofpathway or GO categories, such as to find out which biologicalprocesses are most affected in a given experiment.Our work has focused on interpreting genes and gene lists bybringing them into the context of a wide variety of experimentalconditions. For example, finding out in which tissues, at what time,and in response to which factors a gene is expressed helpsunderstanding the function of this gene and its potential role inone's own experiment. To achieve this, we manually curated,annotated, quality controlled and normalized more than 55'000expression microarrays from thousands of public experiments, andcreated user-friendly tools to visualize gene expression acrossthese contexts. Several such meta-analysis tools are freelyavailable at www.genevestigator.com

Stratified error in the qPCR assays from the statisticalpoint of view

Ales Tichopad

Academy of Science of Czech Republic, Czech Republic

Statistical error affects quantitative PCR at various steps ofsample processing as well as during preparation of a standardmaterial. Quantification efficiency is most commonly evaluatedfrom the slope of standard curve serial concentrations projectedagainst the cycle of quantification. The efficiency is consideredcontributing information to accurately calculate gene expressionrelative to reference sample and/or reference transcript. Yet, whatis the reproducibility of the amplification efficiency estimation in anexperimental setup commonly used in labs? Experiment designwith repeated preparation of the standard curve was used toestimate the uncertainty associated with estimation ofamplification efficiency and its impact on the gene regulation factorcalculated. We show that the additive error component due toamplification efficiency estimation may deter the overall generegulation factor significantly. In addition, we show how varioussample processing steps affect the uncertainty of the regulationfactor.

Primer3: Improvements for the design of qPCRprimers

Andreas Untergasser1, Ioana Cutcutache2, Steve Rozen2

1University of Heidelberg, Germany; 2Duke-NUS GraduateMedical School, Singapore

For over one decade Primer3 assisted scientists in the process ofprimer selection. The popularity of the Primer3 software in thescientific community is indicated by over 5000 citations of theinitial publication. Several software tools employ Primer3 for theprimary selection of primers and reevaluate the output accordingto their special needs, like the tool primer-blast at NCBI. Primer-blast uses Primer3 for the selection of primers and evaluates theirspecificity by blasting them against one of the NCBI nucleotidedatabases. We think the success of Primer3 is based on theaccuracy of primer selection, the fast and stable implementation,the flexibility of the primer selection process and the open sourcelicense which allows the free use, modification and distribution ofPrimer3. Users of Primer3 fall into three groups: bioinformaticians,who embed Primer3 within their web servers, bioinformaticsscripts or pipelines, expert users who select large numbers ofprimers and need tight control over the selection process, andoccasional users who simply want a convenient way to design afew primer pairs. Bioinformaticians usually rely on the Primer3command-line interface and run Primer3 on a local machine(download at sourceforge.net/projects/primer3/). Expert oroccasional users usually rely on the Primer3 web interface,Primer3plus (www.primer3plus.com/cgi-bin/dev/primer3plus.cgi).The functionality of Primer3 has expanded in the last decadeconsiderably since it was first released. Primer3 now offers acleaner interface on the command line, improved primer selectionalgorithms and up to date melting temperature calculations as wellas thermodynamic secondary structure calculations. The primaryfunction of Primer3 is the selection of primers for the amplificationa given sequence. The latest version can also pick lists of primerswhich are not matched into primer pairs, primers required forSanger-sequencing, cloning primers, and can reevaluate user-specified primer pairs. The old algorithm had the tendency to overrepresent very good primers in the result list and to favor certainregions. To overcome this limitation, a region around a selectedprimer can be defined, in which no second primer is allowed tobind and thereby creating a list of truly unique primer pairs whichare well distributed over the sequence. In qPCR applications it isdesired to discriminate DNA derived from mRNA from genomicDNA by using primers spanning an exon-exon junction. Primer3can be provided with a list of junctions and will ensure that eitherthe forward or the reverse primer span one of these junctions.Primer3 is a complex tool, that allows control of the selectionprocess with many parameters. Primer3 now has the option tosave settings for these parameters in a file, enabling expert usersto easily provide optimal parameter settings to less experiencedusers. We are confident that the new modifications positionPrimer3 to be a valuable for the coming decade.


Is normalisation of raw data necessary? TrulyAutomated Analysis of qPCR Data Using theAzurePCR Method

David Kennard, Ze'ev Russak

Azure PCR Limited, United Kingdom

Taking on molecular diagnostics industry challenges of interpretingcycle threshold, creating normalised curves and using 'prettypictures' to facilitate manual interpretation of results, Azure PCRwill demonstrate a novel and unique externally validated methodfor automated analysis of real-time (qPCR) data, includingclassification and quantification. Unlike existing software includedwithin PCR cyclers, the AzurePCR (TM) automated analysismethod is assumption-free and does not require setting of userparameters and, thus delivering time savings for both theresearcher and the clinician. This automated analysis methoddemonstrates close to 100% and even 100% accuracy ofdetection, as confirmed by recent validation studies, including fordata sets with high levels of background noise. We will addresscurrent industry failings by comparing our results against those ofanalysis software packages bundled with popular PCR cyclers.This will be done using recent empirical validations of theAzurePCR automated analysis method conducted with well-knownresearch hospitals and commercial laboratories.

Amplification efficiency as a function of primer andcDNA concentration

Jan M Ruijter1, Quinn D Gunst1, Peter Lorenz2, Maurice JBvandenHoff1

1Academic Medical Center, Amsterdam, Netherlands, The;2University of Rostock, Rostock, Germany

Quantitative PCR data analysis results in strongly biased resultswhen the PCR efficiency is assumed to be 2 (100% efficient) orwhen PCR efficiencies of target and reference genes are assumedto be equal. The amplification efficiency (E) of a single PCRreaction can be derived from the slope of the log-linear part ofindividual amplification curves. The target quantity of theamplicon-of-interest can then be calculated as N0 = Nq / E ^ Cq(in which Nq is the quantification threshold). It has been shownthat the average of the individual PCR efficiencies per amplicongives an accurate and precise estimate of the target quantity persample. However, the causes for the observation of variable PCRefficiencies per amplicon remain largely obscure. Optimizing theprimer concentration in a Sybr Green assay shows that decreaseof the primer concentration results in a decreasing observed PCRefficiency. When the target quantities are calculated for thesesamples, using the mean PCR efficiency per primer concentration,the resulting N0 values are mostly correct. So, for part of therange of primer concentrations the relation between observedPCR efficiency and observed Cq value, Cq = {Log(Nq) - Log(N0)} /Log(E), guarantees that the observed target quantity remainsunbiased. This also shows that in this range of primerconcentrations the PCR efficiency is a constant value from cycle 1onwards. However, when the primer concentration is too low, thePCR efficiency - Cq relation does not compensate for the resultinglow PCR efficiency and a significantly higher N0 value isobserved. A similar dependency of PCR efficiencies and Cqvalues can be observed for the cDNA input concentration. Thisshows that per qPCR assay the primer concentration should beoptimized for the range of target quantities that is expected to bepresent in the samples. Moreover, samples with deviatingindividual PCR efficiencies should be scrutinized when they resultin very high target quantities and, therefore, may have beensubjected to relatively low primer concentrations.

Statistical issues to consider when using qPCR tovalidate microarray or RNA-Seq results.

Zaneta Anne Park

AgResearch, New Zealand

This presentation discusses statistical issues to consider whenrunning a qPCR experiment. These include the importance ofreplication and randomization of treatments on each plate, andusing an experimental design to allocate samples so that thetreatments are balanced across runs. The importance of thesefactors is emphasized by the presentation of data from auniformity trial. We also discuss statistical issues to consider whencreating standard curves. Additionally we present a novel analysismethod in which a new variable which incorporates the efficiency

of each gene is first calculated, and then the data analysed usingan Analysis of Variance. We have found this method easy toimplement and also useful for identifying outliers. A comparison ofthe results using this method and those from REST1 arepresented.1 Pfaffl, M et al. 2002. Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results inreal-time PCR. Nucleic Acids Research, Vol 30, no. 9

Evaluation and applicability of advanced/exotic qPCRquantification strategies and their implementation

Andrej-Nikolai Spiess

University Hospital Hamburg-Eppendorf, Germany

Over its now three years of existence and development, the 'qpcR'package for the R statistical environment has matured into aquantitative real-time PCR (qPCR) data analysis software thathouses nearly 70 functions and example datasets assisting inqPCR analysis. The versatile R statistical language makes itrelatively effortless to implement published algorithms or newideas in a short time, and test these new algortihms by the 20included datasets derived from many different PCR platforms. Thepackage includes in its current version (1.3-4) many ‘replications’of widely-used methods (i.e. window-of-linearity, REST approach,exponential fitting, sigmoidal fitting, threshold cycle estimation bysecond derivative maximum, Cy0, comparative quantitation, ratiocalculation in a batch format, etc). All of these methods have beencompared with the original versions, as to ensure resultcompatibility/identity. Many implemented functions have also beenofficially approved by the original authors. The talk will focus onsomewhat exotic implementations of qPCR quantitation that arerelatively new. While some of them are already published (but notnecessarily available in a software), we will introduce methods thatare still under development or amenable to public discussion inrespect to their general applicability within the qPCR community.The methods that will be presented with short examples are:

1) Monte-Carlo simulation of error propagation (short).

2) Bootstrapping qPCR fits.

3) The development of a tilted threshold, which gives significantlybetter calibration values from high dilution line by an iterativeprocess.

4) A six-parameter sigmoidal model which models the baselineslope accurately.

5) The MAK2/MAK3 mechanistic models from Boggy et al.

6) The maxRatio method from Shain et al.

7) Several methods for automatic tagging of unsuccessful runs.

8) Using the PRESS statistic for goodness-of-fit.

9) The exceptional speed of R when analyzing high-throughputdata. We show this this on a dataset with 9000 runs (rawfluorescence data), whose import, model fitting, quality control(failed runs detection), threshold cycle and efficiency calculationtakes about 10 minutes.

10) The importance of weighting when fitting nonlinear models toqPCR data.

Some these methods will be presented as short animations, toclarify things. We will also show the feasibility of the methods bytesting them on a cohort of datasets.

High-resolution melting data error evaluation betweenruns.

Maksim Bratchikov, Mykolas Mauricas

Centre for Innovative Medicine, State Research Institute, Vilnius,Lithuania

High-resolution melting (HRM) based method for the bacterial(Salmonella spp. as a target) genome typing was applied. Thetargets for this typing were chosen from different variablesequences. All raw data was derived on Rotor-Gene 65H0-100.Slight but essential HRM variations between runs were found forsame samples tested. Therefore, reference samples for this HRMerror evaluation between runs were included. The calculations wesuggested were applied for raw data error normalization betweenruns and fluorescent normalization. We successfully normalizedmelting errors between runs. Data of all samples tested can bestored for the comparison with newly tested samples.


GENEX: data analysis lunch seminar

Time: Wednesday, 30/03/2011: 1:00pm - 2:00pmLocation: Lecture hall 15Session Chair: Mikael Kubista

Amin Forootan

Presentation of GenEx 5.3; the most user friendly andpowerful qPCR experimental design, data analysis anddata mining software yet released!

Mikael Kubista

TATAA BIOCENTER, Sweden

qPCR is today the preferred method for quantitative nucleic acidanalysis, and is being extensively used in routine as well asresearch. Proper usage of qPCR requires careful planning of theexperiment including optimized design, followed by elimination ofsystematic noise by proper handling of controls and references,and mining of the data. This complex flow is made easy withGenEx 5.3. With GenEx a small pilot study is easily planned thatwill identify the sources of confounding variation to the data, aidingthe design of the main experiment and estimating the number ofsubjects needed to secure sufficient statistical power. Themeasured data including relevant experimental settings andsample/assay information are then imported directly into GenExfrom Roche, Agilent, Eppendorf, Bio-Rad, Life Technologies,Illumina and Fluidigm qPCR instruments eliminating the risk ofintroducing error by manually entering experimental parameters,where noise is reduced by proper normalization with standardsand references, and prepared for statistical analysis. Then, withfew clicks with the mouse on user friendly and clear panels,groups are compared using statistical tests, or patterns revealedbased on powerful classification tools. All results are presented inclear and intuitive high resolved graphs ready for publication.

http://genex.gene-quantification.info

MIQE: state of the art & open discussion

Time: Wednesday, 30/03/2011: 2:00pm - 3:00pmLocation: Lecture hall 15Session Chair: Stephen Andrew Bustin

Jo Vandesompele

RDML: state of the art & open discussion

Time: Wednesday, 30/03/2011: 3:30pm – 4:30pmLocation: Lecture hall 15Session Chair: Jan Hellemans

Andreas Untergasser


Abstracts - Poster presentations

Location: Foyer - lower levelTime: Monday, 28/03/2011: 6:00pm - 9:00pm

Tuesday 29/03/2011: 12:00pm - 2:00pmWednesday 30/03/2011: 12:00pm - 2:00pm

All poster sessions will take place in parallel and will last from the"Monday Evening Poster Session" till "Wednesday Lunch PosterSession".

Please put on your poster on Sunday afternoon or Mondaymorning (until noon), and remove it on Wednesday afternoon.Please bring your own drawing pins. You can offer handouts at theposter site.

Poster number Poster session

P001 – P046 New qPCR Applications: MolecularDiagnostics & Expression Profiling

P047 – P055 New qPCR Applications: Method Optimisation& Standardisation

P056 – P061 qPCR Biostatistics & BioinformaticsP062 – P069 High Throughput qPCR & digital PCR Next

Genartion SequencingP070 – P075 Single-cell qPCR qPCR from limited materialP076 – P084 HRM & Methylation StudiesP085 – P090 RNAi: microRNA – siRNA ApplicationsP091 Late submission

New qPCR Applications:Molecular Diagnostics & Expression Profiling

P001

PCR applications for diagnosis in Animal Health

Jovita Fernandez-Pinero1, Montserrat Agüero2

1Centro de Investigación en Sanidad Animal (CISA-INIA),Valdeolmos, Spain; 2Laboratorio Central de Veterinaria (LCV,MARM), Algete, Spain

Viral infectious diseases are a constant risk for livestockproduction due to the economic and sanitary costs produced bytheir entrance into a country. Once the causative agent isidentified, specific strict sanitary measures must be rapidlyimplemented to avoid its spread, and to control and eradicate thedisease. Classical laboratory diagnosis of viral diseases includesboth direct and indirect tools, virus isolation being the “gold-standard” technique to confirm the presence of an etiologicalagent. Around 15 years ago, the merging of the PCR completelytransformed the diagnostic pathway in the National ReferenceLaboratories and soon became an essential tool. Many benefitsprompted its incorporation into routine lab work, high sensitivity,superior to virus isolation, being of most value as allows the earlydetection of pathogens even before the evidence of clinical signsin infected animals. The high specificity, the analysis of a greatnumber of any kind of clinical samples within hours, and thechance to test several pathogens in the same reaction (multiplexassays), made PCR a basic tool for screening diagnostic step.The introduction of the real-time PCR changed the workflow onceagain in lab diagnosis, by reducing analysis time andcontamination risk, and increasing the sensitivity and applicationsin diagnosis. Moreover, real-time PCR accompanied by theappearance of robots in the market for sample preparation andnucleic acid extraction steps, made real a completely automatedanalysis procedure for a high throughput application. Now, PCRhas a wide range of main applications in Animal Health. It isextensively used in surveillance, control and eradication programsof the major viral diseases affecting livestock at national andinternational levels. PCR is a valuable tool in the prevention ofnew disease entrance, the control of biological products, or theevaluation of vaccines efficacy. Generic PCR assays can bedeveloped for detection of new viruses within a genus/family or todetermine the subtype/serotype of the identified virus. Also, PCR

methods can be designed to be used as DIVA (“differentiatinginfected from vaccinated animals”) tests, discriminating vaccinestrains from field circulating viruses, which can be of greatsignificance when using live attenuated vaccines. Finally, PCR isthe starting point in molecular characterization and epidemiologystudies of circulating viruses, essential for tracing the origin andevolution of a disease outbreak. More recently, portable PCRmachines and other simple low-cost PCR-based techniques arebeing launched for on-site application, meaning the analysis ofsamples can be performed in regional labs or even in thefarm/slaughter-house with basic equipment by non-specializedpersonnel. Due to many viral animal diseases are prone to veryrapid spread and the identification of the involved virus is urgentlyrequired, these new tools may become useful first line pen-sidetests in a short time

P002

A Comparative qPCR Analysis of Apoptotic Genes inMilk Somatic Cells of Cows with history ofInconsistent Lactations

Nishant Varshney1, Surender Singh1, Manu Jamwal1,Sudarshan Kumar1, Dhruba Malakar1, Jai Kumar Kaushik1,Sunita Grover1, B S Prakash1, Bishnu P Mishra2, TapasMukhopadhyay3, Ajay K Dang1, Ashok K Mohanty1

1National Dairy Research Institute, Karnal, India; 2National Bureauof Animal Genetic Resources, Karnal, India; 3National Centre forHuman Genome Studies and Research, Punjab University,Chandigarh, India

Milk yield has been a major selection criterion for geneticimprovement in livestock. The milk producing ability of farmanimals differ significantly, which may be due to the expression ofdifferent various signaling proteins, transcription factors, cellsurvival and death factors in the lactating mammary gland. It hasbeen observed that indigenous cows undergo self-induced dryphase earlier than their average lactation period. A change inmammary cells number is a principal cause of decliningproduction. The rate of decline in milk yield with stage of lactationis strongly influenced by the rate of apoprosis. In the presentstudy, we have analyzed the differential expression of variousapoptotic genes such as BCL2, BAD, BAX, STAT3, STAT5,CASPASE3 and CASPASE9 in indigenous cows (Sahiwal breed)between normal lactating vs. short lactating cows. We have usedthe milk somatic cells to ascertain our hypothesis that elevatedexpression of apoptotic genes may lead to short lactation inlactating cows. Short lactating cows taken for the experiment hada lactation length of 100-150 days and the milk yield decreasedsubstantially after this period. Five cows (Sahiwal cows) of 3rd

parity each and having history of short lactation and normallactation length were selected for the experiments. RNA wasisolated from milk somatic cells at different stages of lactationsuch as early, peak, mid and late lactation stages. Fornormalization of qPCR data, two housekeeping genes such asPPP1R11 and β actin were taken as reference genes. Tounderstand the relative pattern of expression of the selectedgenes, expression profile at peak lactation was taken as thecalibrator. After normalization, we observed that all the candidategenes in short lactating cows exhibited elevated expression incomparision to normal lactating cows. Expression level of pro-apoptotic factors such as BAD, BAX, STAT3, Caspase 3, Caspase9 showed fold increase of 18.75, 22.28, 5.8, 5.6 and 4.6 timesrespectively during early lactation and 16.54, 32.8, 5.1, 1.7 and2.6 times respectively during mid lactation in short lactating cowsin comparision to normal lactating cows. Similar trend wasobserved during late lactation. The level of pro-apoptotic Bcl2exhibited 5.4 fold increase during early lactation in short lactatingcows and was subsequently unchanged during mid lactation anddecreased during late lactation. The level of STAT5 exhibited ahigher expression level of 4.6 to 7.1 fold in short lactating cows. Ahigh degree of up-regulation of approximately 17, 32, 5, 2 and 3fold respectively of pro-apoptotic molecules BAD, BAX, STAT3,CASPASE3 and CASPASE9 was observed in short lactatinganimals at the time of abrupt decline in milk yield mimicking theinitiation of accelerated apoptosis. Higher expression of keyapoptotic factors during early lactation in short lactating animalsindicated early initiation of apoptosis leading to untimely cessationof lactation.


P003

Constitutive expression of HSPA-1A gene in twodifferent sources of buffalo (Bubalus bubalis) embryos

Nath Amar, Saikumar G., Gade Nitin Eknath, Taru Sharma G.

Indian Vetrinary Research Institute, India

Among heat shock proteins (HSPs), HSP70 is a most abundantand highly conserved protein which serves as molecularchaperone and provides thermal tolerance. In this study, wedetermined the transcript levels of stress-responsive, heat shockprotein 70.1 (HSPA1A) gene quantitatively by real-time RT-PCR(RT-qPCR) in preimplantation developmental stages of in vitroproduced (IVP) and in vivo derived (IVD) buffalo embryos. Theresults of RT-qPCR revealed that the transcript for HSPA1AmRNA is constitutively expressed in oocytes, the preimplantationdevelopmental stages; 2-4 cell, 8-16 cell, morula and day 7blastocyst of IVD embryos along with their counterpart IVPembryos. Marked differences in the mRNA level was observed at2-4 cell, 8-16 cell, morula and day 7 blastocyst stages betweenthe two embryo sources (IVD and IVP). Amongst IVP buffaloembryos day 7 blastocyst (5764±0.29) had lower HSPA1Atranscript copies as compared to 2-4 cell (9278 ±0.12), 8-16 cell(10849±0.14), and morula (9154±0.23). Although similar trend wasobserved for IVD embryo stages but there was markeddecreament in transcript level; 2-4 cell (7865±0.34), 8-16 cell(10023±0.26), morula (8014±0.18) and day 7 blastocyst(4912±0.36) as compared to IVP embryos. It is concluded thatHSPA1A is synthesized and expressed in stage specific mannerthroughout preimplantation development of buffalo embryosderived in-vivo and in-vitro. It supports the hypothesis thatHSPA1A is synthesized to overcome cellular stress during in vitroculture environment and IVP embryos express high levels ofHSPA1A compared to IVD embryos.

P004

Applicability of qPCR to ensure the quality of milk anddairy products

C LEMAITRE, E LABBE, T RIBEIRO, A CHANGO, A ABDELNOUR

LaSalle Beauvais, France

In food processing industry, quality is an unavoidable element,much for consumers’ health as for organoleptic quality of theproducts. Milk processing is highly concerned because milk issubjected to various contamination sources, before milking (soil,food, water or excrement), during milking (milking people) andafter milking (storage, transport or processing). As qPCR isbooming, we wondered if it was applicable to ensure the quality ofmilk and dairy products. In this view, we did a systematic review ofarticles and reference works published since 1999 dealing withthese problems. We identified several pathogenic and spoilingmicroorganisms such as Staphylococcus aureus, Escherichia coli,Pseudomonas fluorescens, Mycobacterium tuberculosis,Aspergillus flavus. qPCR also allows the quantification ofadulteration e.g. cow’s milk in goat’s milk, and could be used todetect Genetically Modified Organisms. It has been proven thatqPCR is a reliable method, that can be used on various stages ofthe process in question (just after milking, before and aftertransport, after industrial processing…).

P005

Expression study of developmental genes in in vitroproduced buffalo (Bubalus bubalis) embryosgenerated by hand-made cloning and in vitrofertilization

SUDEEPTA KUMAR PANDA, KARN PRATAP SINGH, AMANGEORGE, AMBIKA PRASSNA SAHA, RUCHI SHRAMA,RADHEY SHAM MANIK, MANMOHAN SINGH CHAUHAN,PRABHAT PALTA, SURESH KUMAR SINGLA

NATIONAL DAIRY RESAERCH INSTITUTE, India

High incidence of developmental abnormalities is the majordrawback of nuclear transfer technique, which might be due toinsufficient reprogramming of somatic cell nuclei in enucleatedoocytes. The aim of this proposed study was to examine andcompare the expression of developmental genes in buffalo

embryos produced by hand-made cloning (HMC) which wascompared with in vitro fertilization (IVF). Total RNA was extractedusing cell to cDNA II kit (Ambion, Austin, TX, USA) from pools ofimmature and in vitro matured oocytes (IVM) (n=20 each) andembryos at the morula and blastocyst stages (n=5 each) for bothIVF and HMC groups for performing RT-PCR. Each experimentwas repeated 5 times. The data was analysed using one wayANOVA after arcsine transformation of percentage values. Thepro apoptotic Bax gene expression was found to be notsignificantly (P<0.01) differed in between morula and blastocyststages of embryo produced by both IVF and HMC, and wasundetectable in IVM oocyte. In another case, anti apoptotic Bcl-xlexpression was not significantly differed (P<0.05) in alldevelopmental stages of embryo produced by IVF, whereas itsexpression in morula was significantly higher (P<0.05) than all thestages of embryos produced by HMC. IVF morula has significantlylower (P<0.05) Bcl-xl expression as compared to HMC morula.These results indicated that All the stages of embryo produced byHMC had significantly higher (P<0.05) Bcl-xl expression ascompared to Bax. The expression of Glut-1 was significantlyhigher (P<0.05) in immature oocytes as compared to IVMoocytes,morula and blastocyst stage in both IVF and HMC. Glut-1expression was significantly higher (P<0.05) in blastocyst stageproduced by HMC in contrast to IVMoocytes, and blastocystsproduced by IVF. Whereas no significant difference was observedin its expression during morula stage embryos produced by bothIVF and HMC. The HSP 70.1 was found to be significantly higher(P<0.05) in immature oocytes compared to that in IVM oocyteswhich in turn, was significantly higher (P<0.05) than that inmorulae and blastocysts produced by IVF as well as by HMC.HSP 70.1 was significantly higher (P<0.05) in morula andblastocyst stage embryos produced by IVF as compared to HMCembryos, which was also undetectable in HMC blastocyst. Theseresults indicate that all developmental genes may be downregulated during embryonic development from immature oocyte toblastocyst stage embryos in HMC as compared to IVF. Theestablishment of diagnostic techniques to determine clonedembryos with the potential to develop into normal calves beforeembryo transfer is crucial for the application of somatic cell NT toanimal husbandry.

P006

Validation of RT-qPCR measurements in primarybovine mammary gland epithelial cells (pbMEC)

Diana Sorg, Anne Potzel, Heinrich H. D. Meyer, EnriqueViturro, Heike Kliem

Technische Universität München, Germany

The inner surface of the bovine mammary gland is covered by alayer of epithelial cells. These mammary gland epithelial cells(MEC) synthesize the ingredients of the milk and secrete it into thelumen. They also function as the first barrier to pathogens thatinfect the udder. Cell lines and primary cultures of these cells areoften used for in vitro studies of inflammation and metabolism inthe udder. The common technique for obtaining these cells iscultivating a piece of udder tissue after biopsy or necropsy.Another technique is extracting primary bovine MEC (pbMEC)from fresh milk. Little is known, however, about the changes ingene expression of these cells in the course of cultivation. In thisstudy the aim was to measure the gene expression from pbMECover the course of the first three passages and to compare cellstaken from tissue (n=4) and from milk (n=3). In addition, the effectof cryopreservation in liquid nitrogen on gene expression wasanalysed. Therefore, 2 reference genes for normalisation (GAPDHand Histon) and 8 target genes (Caspase 3, Cytokeratin 8,HMGCR, IL1B, κ-Casein, RANTES, SREBP2 and TLR2) weremeasured. None of the studied factors (cell origin, passagenumber, freezing) had any significant effect on gene expressionlevels (p >0.05). pbMEC extraction from milk is non-invasive andrepeatable. These are advantages in comparison to tissuesampling. Our results suggest that these cells can be equallytaken from both origins regarding the effects on gene expressionin cell culture. Also they can be used during the first threepassages without significant alteration of the gene expressionpattern. Cryopreservation in liquid nitrogen is often necessarypreparing an in vitro experiment. This can also be done withoutinfluencing gene expression of the pbMEC.


P007

Heterogeneity within the pluripotent stem cell state isassociated with transient changes in gene expression

Janice K Au-Young1, Jason Gioia1, Srividya Dadi1, DavidKeys1, Paul J Gokhale2, Peter Andrews2

1Life Technologies, United States of America; 2Centre for StemCell Biology, University of Sheffield, UK

Insight into the molecular, surface marker and epigenetic profilethat makes up the pluripotent state of embryonic stem cells fromboth the laboratory mouse and humans has been gained recently.Previous studies showed that human embryonic stem cell (hESC)

cultures are made up of heterogeneous populations that arepositive or negative for the cell surface marker SSEA3. SSEA3positive cells have a higher percentage of colony formation andhigher levels of pluripotency markers than SSEA3 negative cells.Moreover, different hESC isolates display different propensities fordifferentiation. One obvious source of variation between cell linesis epigenetic differences caused by establishment in culture andprolonged passage. However a more subtle ‘intrinsic’ form ofheterogeneity may exist, wherein a given cell compartmentdefined by a surface marker, has differences in gene expressionand thus differentiation potential. Our studies of hESCs at thesingle cell level provide further support for a model in whichundifferentiated cells can oscillate between cell states within amore general stem cell compartment which is associated withtransient changes in gene expression. Upon culture adaptation,a lower frequency of cells express lineage specific transcriptscompared to normal hESC cultures. Moreover, functional testingfor cells undergoing spontaneous differentiation showed thatadapted hES cell populations are less likely to have endodermalmarkers compared to normal cultures.

P008

Impact of long-term expansion on mesenchymalstromal cell differentiation potential

Andrea Hecker, Irena Brinkmann, Karen Bieback

Institute of Transfusion Medicine and Immunology, MedicalFaculty Mannheim

Mesenchymal stromal cells (MSCs) are promising candidates fornovel cell therapeutic applications. To obtain a clinically relevantdose, expansion seems to be required. Long-term culture,however, can induce spontaneous transformation. Thus weinvestigated whether long-term expansion affects MSCs qualitiesand analysed MSC differentiation capacities by histochemistry,colorimetric assays and reverse transcription quantitative PCR(RT-qPCR). For an exact comparison of mRNA transcriptionconform to MIQE guidelines, RT-qPCR includes internal standardssuch as reference genes (RG) to normalise mRNA levels betweendifferent samples. Classical RG however are deregulated bydifferentiation processes and thus may lead to unreliable results.For accurate RT-qPCR expression profiling of differentiationmarkers, we used geNorm software to determine the most stableRG from a set of five reference genes. Each three osteogenic andadipogenic induced and corresponding noninduced MSCssamples were measured by qPCR and the data analysed by thegeNorm software. For each primer pair the PCR efficiency wascalculated by a standard curve derived of a pool of differentiatedand undifferentiated MSCs. Interestingly, different RG sets wereidentified for adipogenic and osteogenic differentiation pathways:GAPDH, B2M and SFRS4 were found to be the most stable genesfor adipogenic and TBP, SFRS4 and B2M for the osteogenic geneexpression. From this set, a normalization factor (NF) wascalculated and used to determine relative gene expression ofosteogenic and adipogenic markers of long-term cultivated MSCs.Our results revealed that MSC undergo replicative aging whichrapidly affects adipogenic differentiation potential. Osteogenicpotential appeared less affected by the culture age of MSCs.Moreover our data underline the necessity to carefully assessappropriate and valid normalisation controls for RT-qPCR.

P009

Standardisation of triplex real time RT-PCR fordetection and quantification of hepatitis E virus

Petra Vasickova, Petr Kralik, Iva Slana, Ivo Pavlik

Veterinary Research Institute, Czech Republic

Hepatitis E virus (HEV) is the main causative agent of hepatitisnon-A, non-B in humans worldwide. The virus is non-envelopedand its genome contains single-stranded, positive-sense RNA 7.2kb in length. According to sequence comparisons andphylogenetic analysis HEV isolates segregate at least into fourmajor genotypes and 24 subtypes. Genotypes I and II arerestricted to humans and often associated to epidemics indeveloping countries with poor sanitation and hygiene. To thecontrary, genotypes III and IV are responsible for sporadic casesof hepatitis E in both developing and industrialized countries.These genotypes were found in samples of human and animalorigin and thus their zoonotic transmission has been considered.Due to the lack of an efficient cell culture system, the mostcommon methods of HEV detection are techniques based onPCR. HEV genome, as of all RNA viruses, is very polymorphicand thus a single locus PCR fails to detect all possible HEVgenomic variants. Therefore the aim of this study was tostandardise a two-tube reverse transcription triplex quantitativereal time PCR (RT-qPCR) for detection and quantification ofhepatitis E virus (HEV) RNA combining amplification of two HEVloci with an internal amplification control (IAC). Czech strainCZswHEV1 (EU117408), which belongs to the most commonsubtype detected in the Czech Republic, was used as a templatefor RNA standards. These RNA standards as well as IAC wereprepared by in vitro transcription and subsequently used forstandardisation of assay. The limit of detection was experimentallydetermined as 10 copies/µl of the RNA standard for bothamplification targets. The presented triplex RT-qPCR assay candetect at least 10 copies of the HEV genome/µl of isolated RNAand allows quantification based on RNA standards. Usage of RNAIAC facilitates monitoring of false negative results. Based on thesedata, the assay represents significant improvement of HEV RNAdetection.

This work was supported by the Ministry of Agriculture (No.MZE0002716202), the Ministry of Education, Youth and Sports of CzechRepublic (No.OC08045 and CZ 1.05/2.1.00/01.0006 AdmireVet) and by ECGrant Cost Action 929 ENVIRONET.

P010

Molecular basis for the impaired adipogenicdifferentiation potential of cord-blood-derivedmesenchymal stromal cells

Irena Brinkmann, Andrea Hecker, Marianna Karagianni, KarenBieback

Institute of Transfusion Medicine and Immunology, Germany

Mesenchymal stromal cells (MSCs) have received considerableattention for their potential role in cell-based regenerative therapy.For their clinical application, a better understanding of thebehaviour of the cells, e.g. MSCs from different tissue sources,including differentiation, proliferation and migration and thedisparities among them is required. MSCs have the ability todifferentiate into different cell types such as bone, cartilage andfat. Cord blood (CB) derived MSCs, in contrast to the MSCsisolated from lipoaspirate (LA) and bone marrow (BM), differ withrespect to low or absent adipogenic but higher osteogenicdifferentiation potential in vitro. Preadipocyte factor-1 (Pref-1,DLK-1) has been shown via reverse transcription quantitative PCR(RT-qPCR) to be contrarily expressed in CB MSCs in comparisonto LA and BM MSCs during adipogenic differentiation. CB plasma,containing high levels of Pref-1, has shown to inhibit adipocytedifferentiation in LA MSCs, indicating a potential role of Pref-1 ininhibition of adipogenesis. Accordingly, we postulated that Pref-1knockdown via siRNA should induce an adipogenic phenotype inCB MSCs. Unexpectedly; it was not possible to detect adipogenicdifferentiation after 21 days of Pref-1 knockdown, neither on RNAlevel nor as lipid vacuoles. Further studies should ascertainwhether Pref-1 protein is still abundant after 21 days of siRNAtreatment to inhibit the adipogenic differentiation in CB MSCs. Inaddition a screening for Pref-1 interaction partners will beperformed to define whether Pref-1 requires specific proteins fortheir inhibitory role in adipogenesis. Our studies have identified


Pref-1 as one candidate to be responsible for the impairedadipogenic differentiation potential of CB MSCs. It needs to beinvestigated if Pref-1 competitively balances the differentiation intothe adipogenic and osteogenic lineage. Thereby a high Pref-1expression, that yields a lower or absent adipogenicdifferentiation, may consequence a higher osteogenicdifferentiation potential.

P011

qPCR assay for detection of human faecalcontamination in food samples

Lotte Bjerrum1, Anna Charlotte Schultz2, Dorthe LauBaggesen2, Anna Krestine Nørgaard1, Aaron Marc Saunders3

1Danish Technological Institute, Denmark; 2National FoodInstitute, Technical University of Denmark; 3Aalborg University,Denmark

Food and drinking water serves frequently as vehicles fortransmission of human enteric viruses like noroviruses.Contamination of produce can take place during production by useof sewage polluted water or inefficient hygienic norms. Currently,there are no routine testing for viruses in foods and water due tothe lack of validated methods and the methods used in researchlaboratories are still too labour-intensive and expensive to beincorporated in the quality control of most food industries. In thisproject we developed at molecular indicator tool to determine thepresence of human faecal pollution in relevant food sources andgrowth environments. A qPCR assay that detects a specificindicator organism, Bacteroides dorei, unambiguously linked tohuman faeces/wastewater was developed. As extraction of RNAand DNA from mollusks, fruits and vegetables can be challenging,effort was put into optimizing the procedures for extraction, so thatboth RNA (norovirus) and DNA (the bacteria) was retrieved asefficient as possible. The optimisations steps as well as qPCRresults of norovirus and B.dorei will be presented. The impact ofthe method and examples of its use in mollusks, raspberries andother contaminated foods will also be presented.

P012

qPCR method to calculate the copy number of IS1111elements in Coxiella burnetii

Matthias Hanczaruk, Anke Stark, Dimitrios Frangoulidis

Bundeswehr Institute of Microbiology, Germany

Coxiella (C.) burnetii, the causative agent of Q fever, is deemed tohave the potential to be used as a biological weapon. Therefore,investigating outbreaks to trace back the source of an infection isof high importance in forensic microbiology. The insertionsequence IS1111, coding for a transposase, is used as a targetregion for diagnostic PCRs, since it is found in multiple copies inC. burnetii genomes. In silico analyses of the 7 annotated genomesequences demonstrated copy numbers ranging from 12 to 52.Chromosomal rearrangements and DNA insertions/deletionsinvolving these transposable elements are responsible forgenomic plasticity, leading to diversity among isolates, and mayhave influence on virulence. Till now, whole genome sequencinghas been the only reliable method to determine exact IS1111 copynumbers. However, this method is laborious, time-consuming,cost-intensive and certainly not suitable to test many C. burnetiiisolates. We established a quantitative duplex real-time PCRassay based on Taqman-technology, allowing the calculation ofIS1111 copy numbers. To this end, a FAM-labelled probe targetsthe multi-copy IS1111 gene, while the single-copy COM1 gene,encoding for an outer membrane protein, is detected in the HEX-channel. We used the previously sequenced strains Nine Mile andHenzerling, with 20 and 47 IS1111 elements, respectively, tocalibrate the assay. In dilution series, we calculated the efficiencyof the primer-probe-combinations and included the results in ourevaluation. We tested a selected panel of 100 C. burnetii isolatesand demonstrated that the number of IS1111 elements can varyfrom 20 to more than 100 copies. It has been assumed that thecopy number of IS1111 elements can be influenced duringmultiple cell culture or animal passages. With the methodpresented here we could prove this thesis and also will have amarker tool, easy to determine, to control inner herd or otherpopulation related genomic variations of Coxiella burnetii strains.

P013

Quality assurance in GMO analysis – A practicalapproach for estimation of measurement uncertaintyand visualisation of quantitative real-time PCR data

Lars Gerdes, Wilhelm Dicke, Ulrich Busch, Sven Pecoraro


Quantification of genetically modified organisms (GMO) in food orfeed samples in routine laboratories is primarily done by relatingthe copy number for a GMO-specific inserted gene with the copynumber of a species-specific reference gene. The ratio gives acopy-based percentage which is then converted into a weight-based percentage of GMO contents specified in official results.The copy numbers for GMO and reference gene are determinedby two separate quantitative real-time PCR assays with standardcurves using TaqMan chemistry [1]. Measurement uncertainty(MU) is an essential characteristic of experimental results. Thereare many comprehensive guides for estimation of MU available[e.g. 2, 3]. Unfortunately, many of these guidelines have a ratherchemistry background and may not therefore be reliably applied toreal-time PCR measurements. Additionally, few publications existthat deal especially with MU in GMO analysis [e.g. 4, 5, 6]. Wecombined from these - often quite theoretical guidelines - apractical approach for appraisalof MU. Uncertainties related toprecision, recovery, inhomogenity, and reference materials wereestimated and combined in an expanded MU with a confidencelevel of 95 %. As quantification becomes more inaccurate withdecreasing GMO contents, we calculated separate uncertaintiesfor the relevant (legal) thresholds of 0.1 %, and 0.9 % GMOcontent. Quality assurance (QA) requires the control of thereaction parameters that form the basis of the measurements. Wedeveloped a set of control charts for visualising the quality controldata of real-time PCR testing. Spreadsheet software-basedmeasurement analysis is combined with semi-automaticdocumentation for QA purposes. The presented approaches havebecome valuable tools in maintaining the high standards at theBavarian Health and Food Safety Authority (LGL), assured byaccreditation according to DIN EN ISO /IEC 17025:2005.

1. European Union Reference Laboratory for GM Food and Feed (EURL-GMFF). http://gmo-crl.jrc.ec.europa.eu/. 2. ISO, GUM: Guide to theexpression of uncertainty in measurement, ISO, 1995. 3. ISO, ISO/TS21748: Guidance for the use of repeatability, reproducibility and truenessestimates in measurement uncertainty estimation, ISO, 2004. 1-30. 4.Burns, M. and H. Valdivia, A procedural approach for the identification ofsources of uncertainty associated with GM quantification and real-timequantitative PCR measurements. Eur Food Res Technol, 2007. 226: 7-18.5. Trapmann, S., et al., Guidance document on measurement uncertaintyfor GMO testing laboratories, JRC, 2007. 1-41. 6. Žel, J., et al., Calculationof measurement uncertainty in quantitative analysis of genetically modifiedorganisms using intermediate precision--a practical approach. J AOAC Int,2007. 90(2): 582-6.

P014

Quantitative Real Time PCR in Friedreich’s Ataxia:implications for diagnosis and clinical trial design.

Giorgia Puorro1, Antonella Antenora1, Angela Marsili1,Alessandra Denaro1, Raffaele Piro1, Pierpaolo Sorrentino1,Chiara Pane1, Alessandra Tessa2, Sergio Cocozza3, FilippoSantorelli2, Giuseppe De Michele1, Alessandro Filla1,Francesco Saccà1

1Dipartimento di Scienze Neurologiche, University Federico II,Naples, Italy; 2Dipartimento Clinico di Neuroscienze e dell'EtàEvolutiva, IRCCS Stella Maris, Pisa, Italy; 3Dipartimento diBiologia Cellulare e Molecolare, University Federico II, Naples,Italy

Background: Friedreich’s ataxia (FRDA) is the most commonhereditary ataxia among caucasians. The molecular defect inFRDA is the trinucleotide GAA expansion in the first intron of theFXN gene, which encodes for frataxin. Aim of the study was toscreen a population of FRDA patients, carriers, and controls forFXN mRNA levels, and to determine the utility of q-PCR as abiomarker and diagnostic tool. Design/methods: We enrolled 24patients with classic FRDA phenotype (cFA), 6 late onset FRDA(LOFA), 5 compound heterozygotes for expansion and pointmutations (pFA; I154F, IVS4+3delA, R165P), 33 healthyexpansion carriers, and 30 healthy controls. DNA was genotypedfor GAA expansion. Total mRNA was extracted from PBMCs andreverse transcribed using a one-step Master Mix. cDNA was


amplified in multiplex and standardized by quantification of HPRT-1 as a reference gene. Relative expression was calculated usinghealthy controls as a reference group using the efficiencycalibrated model. Analysis was performed with the improvedversion of the relative expression software tool (REST 2009).Continuous variables were analyzed using the one-way ANOVA.Post-hoc analysis was performed with the Bonferroni multiplecomparison’s test. Diagnostic efficacy of FXN mRNA dosage wasassessed constructing ROC curves. Correlation analysis wasperformed calculating Pearson’s coefficient. P values of less that0.05 were considered statistically significant. Results: In cFA,mRNA was profoundly reduced to 19.4% of controls (range 0.06-0.48, p<0.0001) whereas there was a less severe down-regulationin pFA (52.7% of controls , p<0.0001), reulting different mRNArelative expression levels between cFA and pFA (p<0.001). LOFApatients and carriers showed a less severe down-regulation to50.4% (range 0.35-0.85, p<0.0001) and 53.0% (range 0.11-1.21,p<0.0001), respectively. Comparison of cFA vs controls, resultedin 100% sensitivity and specificity for a cut-off frataxin mRNAvalue of 0.6 as compared to controls (p<0.0001, area=1.00).Comparison of pFA with controls resulted in a sensitivity of 100%and specificity of 88% for a cut-off frataxin mRNA value of 0.75(p<0.001, area=0.976). For mRNA correlation analysis, cFA andLOFA were considered together as a single group. mRNA levelscorrelated directly with age at onset (P<0.001, R2=0.3905) andinversely with GAA1 and GAA2 (p<0.01, R2=0.2364; p<0.05,R2=0.1750). We found an inverse correlation between mRNAlevels and GAA2 in carriers (p<0.0001, R2=0.5011). Conclusion:We report the first explorative study on frataxin mRNA levels inPBMCs from a cohort of FRDA patients, carriers and healthycontrols. FRDA patients showed reduced levels of FXN mRNA toone-fith of control levels. Messanger RNA levels proved to bediagnostic when comparing cFA to controls with 100% sensitivityand specificity. In contrast, q-PCR is not able to differentiate pFAand controls. Clinical trials should be designed to include FXNmRNA levels as an endpoint.

P015

Cutaneous Mycobacterium marinum infection inhumans detected by quantitative real time PCR

Michal Slany1, Krystyna Kantorova1, Petr Jezek2, MonikaBodnarova3, Ivo Pavlik1

1Veterinary research institute, Czech Republic; 2County HospitalPribram, Czech Republic; 3Charles University Praque, FirstFaculty of Medicine and General University Hospital, CzechRepublic

The low frequency of infection caused by non-tuberculousmycobacteria (NTM), non specific symptoms for individual NTM,requirements of specific cultivation approaches, very often are notroutinely used microbiological laboratories and the lack of specificidentification methods could detract from a correct diagnosis.Mycobacterium marinum is occasionally associated withcutaneous infections in humans so called “fish tank granuloma”.Rapid, sensitive and specific method for the detection ofM. marinum in animal and human samples was set up in thisstudy. The duplex real-time PCR assays (erp) described hereprovides a fast, sensitive and specific diagnosis of M. marinum, incomparison to more time-consuming conventional PCR and DNAhybridization methods. We developed a quick and sufficientlysensitive system for the detection of M. marinum in tissue sampleswith a clinical sensitivity of 5 × 102 CFU per 50 mg of tissue for erpqPCR and with median of isolation yield 57.79%. DevelopedqPCR system was successfully used for detection of M. marinuminfection in two humans. Interestingly, one of the patients waspreviously misdiagnosed and treated with methylprednisolone forperiod of 3 month, which resulted in a rare systemic spread ofM. marinum into the testis and epidimidis. Simultaneously, thescreening for M. marinum presence in both patients’ aquariumenvironments revealed infected fish with M. marinum. The protocolused enabled us to complete analysis of sample, includingcontrols, in approximately 6 hrs; including extraction and assaytime, and is suitable for implementation in routine laboratorydiagnostics. To our best knowledge this is the first report ofdetection and quantification of M. marinum directly from infectedhuman tissue.

This work was supported by Grants Nos. MZE0002716202 and QH91240from the Ministry of Agriculture and Grant “AdmireVet” No. CZ1.05/2.1.00/01.0006-ED0006/01/01 from the Ministry of Education, Youthand Sports of the Czech Republic.

P016

Quantization of Rauscher-like MuLV in plasma fromBALB/cJ mice

Yehuda Stram1, Marisol Rubinstein1, Evgenia Lubashevsky1,Sofa Savranski1, Gavriel Leitner1, Zeev Treinin2

1Kimron Veterinary Institute, Israel; 2Israel Dairy Board

Recently it was reported by Ter-Grigorov , et al., 1997 that a newvirus arose in BALB/c females which were repeatedly mated toC57BL/6 (B6) males, and then injected with fixed, activated B6male spleen cells. The clinical symptoms of the infected mice, bythe new virus, resembled those of AIDS in man. To characterizethe virus, BALB/cJ mice were inoculated with virus-containingplasma which was derived from mice 3 weeks post infection.Biological and molecular analysis of the new virus revealed anovel virus mixture of murine leukemia viruses (MuLVs), termedRauscher-like MuLV, that causes an increase in hematopoiesisdue to activation of pluripotent HSC. It could be demonstrated thatendogenous BALB/c mouse ecotropic and xenotropic MuLVs areactivated by these treatments. multiple pregnancies and allostimuliappear to have provided the signals required for activation of andrecombination among endogenous viruses and could haveresulted in generation of the Rauscher-like MuLV mixture. Tobetter understand the phenomena we aimed to determine theamount of each of the viruses in the mixture. Taqman technologywas employed for this purpose. To establish standards for eachvirus measurements, fragments from each of the viruses carryingthe Taqman amplicon were amplified in a way that the forwardedprimer carried the T7 promoter sequences. Each of the amplifiedfragments were transcribed using T7 RNA polymerase. Ten folddilution of the newly transcribed fragment were used to establishstandard curves. It could be shown that about 10 viral RNAmolecules per sample could be detected using this system. Thedeveloped technique will be used to study the underlinedmolecular processes that leads to the production of this uniqueviral mixture and its role in aids like disease in the affected mice

P017

Development of a real-time quantitative multiplexTaqMan-based RT-PCR assay for detection andquantification of measles, mumps and rubella viruses

Yulia Zabiyaka, Eugeny Faizuloev, Sergey Lobodanov,Tatiana Konstantinovna Borisova

Mechnikov Research Institute of Vaccines and Sera, RAMS,Russian Federation

Vaccine potency is a major property for the quality of live virusvaccines. Potency assay of trivalent measles, mumps and rubella(MMR) live virus attenuated vaccine is performed routinely byplaque or conventional 50% cell culture infective dose (CCID50)assays. However, these assays are time-consuming, labor-intensive, typically highly variable and requiring the availability ofhighly specific neutralizing antibodies in order to allow detection ofthe infectivity of the individual components in multivalent vaccines.

In the present study, novel methods to estimate the potency ofMMR viruses in virus-containing bulks using real-time quantitativeTaqMan-based reverse-transcription PCR (qPCR-RT) have beendeveloped for each virus and the principal possibility of MMR virusquantification using Multiplex assay format has been shown.Specific primers and probes were designed and the qPCR-RT wasoptimized by annealing temperature and MgCl2 and primerconcentration. The results determined by qPCR-RT method havebeen compared in parallel with that of a conventionalCCID50 assay for MMR virus samples obtained daily during long-term cultivation and for the International Reference Reagents ofMMR vaccines (Live). For potency estimation by Multiplex qPCR-RT and CCID50 assay laboratory variant of trivalent MMR vaccinecontaining the L-16 measles, the L-3 mumps and the Wistar RA27/3 rubella virus strains and commercial batches of MM divalent(Microgen, Russia) and MMR vaccines (Priorix, GSK Biologicals,Belgium) were used. The potency of the viruses determined byqPCR-RT was estimated relative to the 10-fold dilutions of thereference samples with titers 5,4, 5,2 and 6,3 lgCCID50/ml forMMR viruses respectively and the corresponding trivalent sample.To control tube-to-tube variations the Human Rhinovirus 16 as aninternal positive reference was used. It was found that the


difference between viral titer measured by qPCR-RT and thecorresponding infectious viral titer measured by CCID50 assay inpaired samples did not exceed 0,3 lgCCID50/ml for certain periodafter inoculation (WHO allowed value) and the Pearson coefficientbetween results obtained by two assays was close to 1, indicatinga significant correlation. For the reference samples of vaccines,the discrepancy between the titers determined by qPCR-RT andCCID50 assays was within 0,2 lgCCID50/ml, which is less thanWHO allowed value. The developed qPCR-RT method was shownto have high efficiency, high sensitivity and high reproducibility.This assay is virus-specific and serological neutralization can beomitted, besides it was faster and less laborious compared to theclassical assay. While the traditional methods would still benecessary to determine viral potency (in term of infectious virus),the novel qPCR-RT was demonstrated to be allowed method forrapid determination of optimal harvest time and bulk virus titers. Italso can be used for rapid detection of MMR viruses in clinicalspecimens.

P018

Comparative study of direct sequencing andTheraScreen assay for EGFR mutational analysis

LUZ MARTINEZ AVILES, JAVIER GIMENO, RAQUELLONGARON, ERICA TORRES, GEMMA NAVARRO, LARAPIJUAN, SERGI SERRANO, BEATRIZ BELLOSILLO

HOSPITAL DEL MAR, Spain

BACKGROUND: Epidermal growth factor receptor tyrosine kinaseinhibitors are used in non-small cell lung cancer (NSCLC).Patients carrying mutations in the EGFR gene have a betterresponse to this therapy than patients with wild type EGFR. AIM:The aim of this study was to compare the analytical sensitivity oftwo different procedures to detect EGFR mutations. METHODS:We have determined the mutational status of the EGFR gene intumoral samples from 200 patients with NSCLC by twomethodologies: 1. PCR amplification of exons 18, 19, 20 and 21 ofthe EGFR gene followed by direct sequencing using BigDye 3.1and analysis in a genetic analyzer (Applied Biosystems) and 2.TheraScreen (DxS) assays using a real time PCR system(Applied Biosystems). RESULTS: Analysis of EGFR mutations in200 biopsies of NSCLC patients by both techniques showed aconcordance of 97%. From the whole cohort, 169 patients werenegative for EGFR mutations whereas 31 patients presentedmutations (at least by one technique). From the group of EGFR-positive patients, in four cases the mutation was only detected bythe TheraScreen assay because the mutant allele burden wasvery low. In two additional cases, the mutation was only detectedby direct sequencing. In the first one the patient carried the V769Mmutation, an alteration that is not included in the commercial kit. Inthe second case the patient harboured the G719S mutation atexon 18 (which is tested in the commercial kit), but also the E709Amutation at the same exon that probably disrupted the binding ofthe probe, so we obtained a false negative result with theTheraScreen assay. CONCLUSIONS: Both direct sequencing andTheraScreen assayshowed a good correlation althoughTheraScreen assay has a greater sensitivity than directsequencing in samples with low mutant allele burden. TheTheraScreen assay may give some false negative results becausesome infrequent mutations are not included in the test andadditional mutations near the ones tested in the kit may affect thebinding of the probes.

P019

Real-time PCR with SYBR Green and melting curveanalysis as a diagnostic screening tool for protozoanoocysts

Laura F. Lalonde, Alvin A. Gajadhar

Canadian Food Inspection Agency, Canada

A simple screening method for the detection and identification ofprotozoan oocysts is a necessary diagnostic tool for human,animal, and food-borne disease outbreak investigations. Ourobjective was to develop a real-time quantitative PCR (qPCR)assay with melting curve analysis (MCA) to detect, differentiate,and identify DNA from protozoan species of veterinary, zoonotic,and food safety importance. We designed and employed auniversal coccidia primer cocktail consisting of seven

oligonucleotides to amplify 18S rDNA from Cryptosporidiumparvum, Toxoplasma gondii, Cyclospora cayetanensis, andseveral species of Eimeria, Sarcocystis, and Isospora using qPCRwith SYBR Green detection. A standard curve was constructedfrom DNA of serial dilutions of 105, 104, 103, 102, or 101T. gondiioocysts to determine assay sensitivity. DNA from as few as 10 T.gondii oocysts could be routinely detected. Melting temperatures(Tm) were determined for each species based on MCA of multiplereplicates over several runs. Tm data analysis showed that C.cayetanensis, C. parvum, Cryptosporidium muris, T. gondii,Eimeria bovis, Eimeria acervulina, Isospora suis, and Sarcocystiscruzi could each be identified by unique melting curves anddifferentiated based on Tm. The qPCR MCA assay successfullyamplified the target gDNA of both C. parvum and C. cayetanensisor S. cruzi and T. gondii when these species were together in amixed template. When used with suitable controls, this qPCRMCA assay could be used to sensitively detect, reliablydifferentiate, and rapidly identify DNA of protozoan oocysts infaecal, food or clinical diagnostic samples using readily availableSYBR Green dye and without the need for a high-resolution MCAqPCR instrument. Further validation of this simple qPCR MCAassay using contaminated field samples and multiple isolates ofeach species will support its application as a routine diagnosticscreening tool for parasitology.

P020

RNA isolation from high-sugar content freeze-driedtomato (Solanum lycopersicum L.) fruits and qRT-PCRanalysis

Libert Brice TONFACK1,2, Emmanuel YOUMBI1, Benoit van-der-Rest2, Alain LATCHE2, Jean-Claude PECH2

1University of Yaoundé I, Cameroon; 2UMR990, Genomic etBiotechnologie des Fruits INRA/INP-ENSAT, Toulouse, France

With minor modifications, we applied a previously reported RNAisolation protocol that used phenol/chloroform/isoamylalcohol tolyophilized (freeze-dried) fruits of tomato (Solanum lycopersicumL.). When gene expression profiles are studied, plant materialmust be preserved in its collected state. Fresh plant materialcannot feasibly be transferred at ultra-low temperatures fromnatural habitats to the laboratory. We explored the use oflyophilized tissue for RNA isolation from two varieties of tomatofruits. High yields of RNA (~236 μg/g dry weight of fruit tissue)were obtained, and the RNA was suitable for all molecular biologymethods tested, including quantitative reverse transcription RealTime Polymerase Chain Reaction (qRT-PCR) analysis. Wedemonstrated that RNA obtained from freeze-dried fruit tissue wasof good quality, undegraded, and useful for all moleculardownstream applications. This work opens an important ways toimprove and sustain researches in conditions of lack of moderntools for better development.

P021

Seasonal quantification of Grapevine fanleaf virus byone-step RT real-time PCR

Urska Cepin, Ion Gutiérrez-Aguirre, Maruša Pompe-Novak,Kristina Gruden, Maja Ravnikar

National Institute of Biology, Slovenia

Grapevine fanleaf virus (GFLV) is a +ssRNA virus, which is thecausal agent of the fanleaf degeneration disease on grapevines.The objective of this study was to design and validate an RTquantitative real-time PCR (RT-qPCR) assay, to efficiently quantifyGFLV virus in grapevine tissues through the whole season. Formethod validation total RNA was isolated from grapevine materialcontaining the reference GFLV isolate F13. LOD and LOQ for thedesigned one-step RT-qPCR assay were evaluated by testing 10-fold serially diluted RNA (from undiluted to 10-8) in triplicate (intra-assay) in three independent runs (inter-assay). The dilutionswhose Cq values from three separate runs gave coefficient ofvariation (CV) ≤ 30% were assumed to be within the range ofquantification (Burns et al., 2004). Theoretical genome copyconcentrations per reaction were assigned, taking into accountthat the manifestation of stochastic effects (not all reactions intriplicate detected or a high intra- or inter-assay CV) is indicative ofthe presence of ≤ 10 target copies per reaction (Ellison et al.,2006). The results showed that reliable quantification could be


performed within the range from 102 to at least 107 genome copiesper reaction. Phloem, which is the toughest grapevine material fordownstream processing and analysis, was sampled from sixgrapevines in Slovenia at four seasonal time (June, July, Augustand September). RNA was isolated and 10-fold and 100-folddilutions tested in duplicates with the RT-qPCR assay for GFLVplus two reference gene assays (COX and 18S). Both referencegenes were compared using geNorm software (Pfaffl, 2001;Vandesompele et al., 2002), obtaining an M value of 0.488. Thisindicates a high seasonal correlation and stability of the COX and18S expression, which, together with the fact that both genesbelong to different metabolic pathways, makes them suitablenormalisers for GFLV relative quantification. Relativequantification was expressed as a ratio (r) of the GFLVconcentration in each sample to the GFLV concentration in adefined calibrator sample and normalised to the reference geneexpression, which was represented by the geometric mean of bothreference genes (Vandesompele et al., 2002). The relativeexpression ratio was calculated based on the amplificationefficiencies of both GFLV and the reference genes, E=10(1/slope)(where the slope means DCq between 10- and 100-fold dilutions)(Pfaffl, 2001). The inclusion of amplification efficiencies togetherwith the reference gene normalisation minimized potentialintersample variations due to differences in the efficiency of RNAisolation, reverse transcription and the amplification itself. The rvalues were proportional to the GFLV genome concentration andshowed seasonal fluctuations of GFLV concentration in phloemwith the lowest amount of viral RNA being found during thesummer in the majority of the tested grapevines.

P022

Submicroscopic duplications identified duringprenatal aneuploidy testing

Alison Hills1, Kathy Mann1, Caroline Mackie Ogilvie2

1GSTS Pathology, Guys and St Thomas NHS Foundation Trust,United Kingdom; 2Cytogenetics Department, Guys and St ThomasNHS Foundation Trust, United Kingdom

Rapid semi-quantitative QF-PCR (Quantitative FluorescencePCR) is a widely-used approach for the prenatal diagnosis ofviable autosomal aneuploidies. Since 2000, we have tested>40,000 prenatal samples using a one-tube multiplex containingseventeen polymorphic microsatellite markers specific forchromosomes 13, 18 and 21 to diagnose trisomy for thesechromosomes. During this period, 67 (~0.15%) samples exhibiteda triallelic profile at a single locus, with all other flanking locishowing normal biallelic or uninformative results. Such findingsmay indicate either benign submicroscopic duplications (SMDs),or partial chromosome imbalance with phenotypic consequences.Five recurring SMD regions were identified on chromosome 13,six on chromosome 18 and three on chromosome 21. In all butone case, QF-PCR analysis of parental blood samples showedinheritance of the SMD, indicating benign variation; however, theadditional testing required to establish this is likely to causeparental anxiety. In view of this, CMGS/ACC Best PracticeGuidelines now recommend that SMDs that have previously beenassociated with a normal parental phenotype are not reported;markers that identified the most frequent of these have beenreplaced in our multiplex. However, previously unreported SMDsthat are not present in the database of genomic variants presentdifficulties of interpretation, especially when found to be de novo,or to represent terminal or unflanked loci. It is therefore importantthat the incidence and aetiology of these SMDs is made available.The inherited and de novo SMDs identified in our laboratory will bepresented. One SMD region on chromosome 13 was investigatedusing quantitative FISH and PCR; the duplicated segment wasfound to be between 20 and 130 kb in length and appeared to betandem and of a similar size in all tested cases.

P023

The Design and Construction of Competitor Fragmentby SOE-PCR for Quantification of The RuminalBacterium: Butyrivibrio fibrisolvens

Amir Taheri Ghahfarokhi, Mojtaba Tahmoorespur, MohammadReza Nassiry, Mohammad Hadi Sekhavati

Ferdowsi University of Mashhad, Mashhad, Iran

Butyrivibrio fibrisolvens strains are presently recognized as themajor butyrate-producing bacteria found in the rumen. They canbe found in the digestive track of many animals and also in thehuman gut. Aim of this study was to develop a powerfulquantitative competitive polymerase chain reaction (QC-PCR)assay based on 16S rDNA for the enumeration of the strainsbelonging to Butyrivibrio fibrisolvens. Species-specific PCRprimers used to amplify partial 16S rDNA region (213 bp as targetDNA) were chosen from the literature. Two internal primers,bearing 5' tails which contain two ~30 nucleotide sequences whichare unrelated to the target to be amplified and complementary toeach other, were designed. To construct the homologouscompetitor with 50 bp insertion a stepwise SOE-PCR (Splicing byOverlap Extension Polymerase Chain Reaction) in three separateamplifications using different primer pairs was carried out. Forseveral future applications and as the easiest way to determinecompetitor concentration, competitor fragment was cloned into aTA plasmid vector (pTZ57R/T) and was purified and quantifiedbefore used. Competitor was serially diluted and co-amplified byPCR with total extracted DNA from rumen fluid samples. QC-PCRproducts were electrophoresis on agarose gel containing ethidiumbromide, and photographed. Band intensities were measuredusing image analysis software (imageJ 1.42q) to determine if co-amplification occurred with equal efficiency. Plotting the quantity ofcompetitor against the ratio of amplified target to amplifiedcompetitor using log scale was evaluated by simple regression byusing JMP software and finally R2 was estimated as a criterion ofcompetitive PCR performance. The log plot of the amount ofamplified target DNA against the amount of amplified competitorDNA was highly linear (R2 = 0.985). As a result of our study a newcompetitive PCR assay was developed for quantification andenumeration of Butyrivibrio fibrisolvens in rumen fluid samples.

P024

A Modular approach in method validation of the real-time PCR module for the detection of five VTECEscherichia coli serogroups

Dafni-Maria Kagkli1, Thomas P. Weber1, Marc Van denBulcke1, Silvia Folloni1, Stefano Morabito2, RosangelaTozzoli2, Monica Ermolli1, Laura Gribaldo1, Guy Van den Eede1

11European Commission Joint Research Centre, Institute forHealth and Consumer Protection, Molecular Biology andGenomics Unit, via E. Fermi 2749, I-21027, Ispra (VA), Italy;2Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161,Rome, Italy

The detection and identification of the presence of pathogenicmicro-organisms in products often requires the simultaneousdemonstration of distinct DNA sequences in a single viableorganism. While the latter can only be demonstrated with classicalmicrobial assays, combined PCR analysis allow for the verysensitive detection of multiple pathogenic sequences in a sample(be it DNA or RNA). To support concise decisions, the appliedPCR methods should however exhibit similar performance for anumber of criteria (Bustin et al., 2009). In various fields wherePCR is applied, the method specificity and sensitivity are generalyrecognized as the minimal critical performance parameters. Here,we demonstrate the results of the assessment of a set of real-timePCR methods proposed to CEN (CEN TC275/WG6) for thedetection of Verocytotoxin Escherichia coli (VTEC). While the ISOstandard 16654:2001 is focused only on the detection of E. coliO157 serogroup, we have extended the scope to a number ofother important serogroups (O26, O103, O111, and O145).Specificity of the methods was demonstrated in silica bybioinformatics analysis and experimentally. The dynamic rangeand the PCR efficiency of all methods was determined bystandard curve dilution. Finally, the limit of detection of eachmethod was determined by serial dilution analysis. All PCRmethods apply TaqMan probe-based detection, targeting the twoE. coli toxin genes, their variants (vtx1, vtx2), and the intimin (eae)gene. All methods were optimized and assessed against thecriteria set by the EURL-GMFF for real-time PCR methods(http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm). All methodsperform adequately: all are highly specific to the expected targets,exhibit a linear correlation between 10 to 10.000 copies, have anLOD ranging from 10 to 20 copies and a PCR efficiency of >90 %(except for the methods for O103 and O145). Our datademonstrate that this set of VTEC detection/identification methodsrepresent a valuable series of PCR methods that can be combined


into a reliable VTEC decision support system. The applied so-called 'modular approach' in the method performance assessmentwill be discussed.

Stephen A. Bustin, Vladimir Benes, Jeremy A. Garson, Jan Hellemans, JimHuggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W. Pfaffl,Gregory L. Shipley, Jo Vandesompele, and Carl T. Wittwer (2009) TheMIQE Guidelines - Minimum Information for Publication of QuantitativeReal-Time PCR Experiments. Clinical Chemistry. 55(4): 611-622

P025

Comparative study of direct sequencing andTheraScreen assay for KRAS mutational analysis

LUZ MARTINEZ AVILES, MAR IGLESIAS, ESTER MORAGON,ERICA TORRES, GEMMA NAVARRO, MAR GARCIA GARCIA,SERGI SERRANO, BEATRIZ BELLOSILLO

HOSPITAL DEL MAR, Spain

BACKGROUND: Epidermal growth factor receptor inhibitortherapy is used for treatment of metastatic colorectal carcinomas(CRC) and non small cell lung cancer (NSCLC) in patients lackingKRAS mutations. Patients carrying mutations in the KRAS genedo not respond to this therapy. AIM: The aim of this study was tocompare the analytical sensitivity of two different procedures todetect KRAS mutations. METHODS: We have determined themutational status of the KRAS gene in tumoral samples from 151patients with CRC and 13 from NSCLC patients by twomethodologies: 1. PCR amplification of exon 2 of the KRAS genefollowed by direct sequencing using BigDye 3.1 and analysis in agenetic analyzer (Applied Biosystems) and 2. TheraScreen (DxS)assays using a real time PCR system (Roche Diagnostics).RESULTS: Analyzing KRAS in 151 biopsies of CRC patients weobserved a 98% of concordance between both techniques. Ninety-nine patients were negative for KRAS mutations in codons 12 or13. Forty-seven patients presented mutations in one of these twocodons, but 6 out of 47 cases showed doubtful sequencingresults, so the TheraScreen assay was determinant to establishthe final positivity of these cases. Moreover, the TheraScreenassay detected three positive cases which direct sequencing hadfailed to detect due to the low mutant allele burden. In addition, weanalysed 13 patients with NSCLC of which 10 showed ambiguoussequencing results so the TheraScreen assay was needed toconfirm the mutations. Finally, four cases presented by directsequencing, infrequent KRAS mutations (G13R, G13C andG12F). The TheraScreen assay was not able to detect the twomutations affecting codon 13 because they are not included in thetest. Regarding the G12F mutation, although it is not included inthe assay, it showed an unspecific positivity for the G12Vmutation. CONCLUSIONS: Both direct sequencing andTheraScreen assayshowed a good correlation althoughTheraScreen assay has a greater sensitivity than directsequencing in samples with low mutant allele burden.TheraScreen assay may be, in some cases, less specific thandirect sequencing and additional mutations should be included inthe test in order to avoid false negative results.

P026

Developing an optimal qRT-PCR strategy for fungaltranscriptomics on carotenoid metabolism

Yamuna Sahadevan1, Wilhelm Boland1, Kerstin Hoffmann2,Kerstin Voigt2

1Dept.of Bioorganic Chemistry, Max Planck Institute for ChemicalEcology,Jena, Germany; 2Jena Microbial Resource Collection,Dept.of Microbiology and Molecular Biology,University of Jena andHans-Knöll-Institute, Jena,Germany

In zygomycetous fungi, β–carotene production is augmentedseveral folds in mated cultures rather than in individual matingpartners. Despite the fact that a cocktail of trisporic acid sexhormones induce carotenogenesis, little is known about the linkbetween these two factors at the genetic level. We aim to developa reliable qRT-PCR method following MIQE guidelines for themRNA quantification of fungal genes involved in carotenoidmetabolic pathway. Mating experiments with Blakeslea trisporawere done for kinetic analysis of 4 target genes using act-1(actin)as the reference gene for relative quantification. A standard curvewas used to determine the reaction efficiency of primers based ona 5-fold dilution series, using in-built Stratagene Mx3000Psoftware. A high annealing temperature of 60° C with 35 cycles

based on SYBR Green chemistry were found to be optimal for theassay. Kinetic studies pinpoint an upregulation in carB and carRAgenes involved in carotene biosynthesis which extends from anearly growth phase of 24hours upto 144hours (6days).Contrarily,the expression pattern of tsp3 and tsp1 involved in β–carotenecatabolism remain stable throughout. For ensuring reliableconclusions, we are investigating 3 more reference genes namely,gpd (Glyceraldehyde phosphate dehydrogenase), EF-1α(translation elongation factor 1-alpha) and pyrG (orotidine- 5’-monophosphate decarboxylase) for data analysis andnormalization using the software Qbase.

P027

Development and evaluation of a one-step real-timeRT-PCR assay for universal detection of influenza Aviruses from avian and mammal species

Alexander Nagy1, Veronika Vostinakova1, ZuzanaPirchanova1, Lenka Cernikova1, Zuzana Dirbakova2, MiroslavMojzis2, Helena Jirincova3, Martina Havlickova3, Adam Dan4,Krisztina Ursu4, Stefan Vilcek5, Vlastimil Krivda1, JitkaHornickova1

1State Veterinary Institute Prague, National Reference Laboratoryfor Avian Influenza and Newcastle Disease, Czech Republic;2State Veterinary Institute Zvolen, National Reference Laboratoryfor Avian Influenza and Newcastle Disease, Zvolen, SlovakRepublic; 3National Institute of Public Health, National ReferenceLaboratory for Human Influenza, Prague, Czech Republic;4Central Agricultural Office, Veterinary Diagnostic Directorate,Molecular Biology Laboratory, Budapest, Hungary; 5University ofVeterinary Medicine in Kosice, Department of Parasitology andInfectious Diseases, Kosice, Slovak Republic

The objective of our study was to develop and evaluate a TaqManreal-time RT-PCR (RRT-PCR) assay for universal detection ofinfluenza A (IA) viruses. The primers and LNA-modifiedoctanucleotide probe were selected to correspond to extremelyconserved regions of the membrane protein (MP) segmentidentified by a comprehensive bioinformatics analysis including10,405 IA viruses MP sequences, i.e., all of the sequences of theInfluenza Virus Sequence database collected as of August 20,2009. The RRT-PCR has a detection limit of approximately fivecopies of target RNA/reaction and excellent reaction parameterstested in four IA viruses reference laboratories. The inclusivity ofthe assay was estimated at both the bioinformatic and theexperimental level. Our results predicted that this RRT-PCR assaywas able to detect 99.5% of known human IA virus strains,99.84% of pandemic influenza A (H1N1) strains, 99.75% of avianstrains, 98.89% of swine strains, 98.15% of equine strains, and100% of influenza A viruses of other origin.

P028

Dietary fibers are not born equal: qPCR to defineprebiotic properties

F DEPEINT, G ABDEL NOUR, CN NIAMBA, P POUILLART, AABDEL NOUR


Prebiotics have been defined by Roberfroid et al (2004) asselectively fermented ingredients that allow specific changes, bothin the composition and activity of the gastrointestinal microflorathat confer benefits upon host well being and health. Mostresearchers consider that changes in the composition of gutmicroflora can be characterised by an increase in probioticbacteria (eg. Bifidobacterium, Lactobacillus) and/or decrease inpathogenic bacteria (eg. Bacteroides, Clostridium). Similarly,changes in the bacteria activity is often characherised by abutyrogenic activity. We present here two clinical trialsinvestigating prebiotic potential of dietary oligosaccharides. Ineach of those studies Bifidobacterium and Clostridium wereamplified using qPCR and quantified against standard curves.


P029

Disrupted balance between phase I and phase IIestrogen-metabolising enzymes may contribute to thegrowth of endometriotic tissue in ovarianendometriosis

Neli Hevir1, Martina Ribič-Pucelj2, Tea Lanišnik Rižner1

1Institute of Biochemistry, Faculty of Medicine, University ofLjubljana, Ljubljana, Slovenia; 2Department of Obstetrics andGynaecology, University Medical Centre, Ljubljana, Slovenia

Endometriosis is a complex estrogen-dependent disease definedas the presence of endometrial tissue outside the uterine cavity.Estrogens stimulate cell proliferation via the estrogen receptor andundergo extensive oxidative metabolism at different positions,catalyzed by various cytochrome P450 isoforms. Formation 2- and4-hydroxy-estrogens (catechol estrogens, CEs), is catalysedmainly by CYP1A1/1A2 and CYP1B1, respectively. CEs can beoxidized to the corresponding estrogen ortho-quinones withconcomitant formation of the reactive oxygen species (ROS).NAD(P)H:quinone oxidoreductase (NQO1/2) regulate thereduction of toxic estrogen quinones back to catechols.Furthermore, estrogens can undergo 16α-hydroxylation,extrahepaticaly catalysed mainly by CYP3A5 and CYP3A7. CEand 16α-hydroxy-estrogens are further metabolised by theconjugative enzymes: catechol-O-methyltransferase (COMT),sulfotransferases (SULTs), UDP glucuronosyltransferases(UGTs), and glutathione S-transferases (GSTs) and yields lessharmful products. Expression levels of genes encoding phase I(CYP1A1, CYP1A2, CYP1B1, CYP3A5, CYP3A7) and phase II(SULT1A1, SULT1E1, SULT2B1, COMT, UGT2B7, NQO1, NQO2,GSTP1) estrogen-metabolizing enzymes were studied by real-timePCR in 31 samples of ovarian endometriosis and 29 normalendometrium samples. PPIA, GAPDH, and RPLP0 were used fornormalisation. MIQE guidelines were considered in theperformance and interpretation of the qPCRs. We foundsignificantly higher levels of CYP1A1 and CYP3A7 (p < 0.0001)suggesting increased 2- and 16α-hydroxylation of estrogens.CYP1B1 and CYP3A5 expression was not altered, while CYP1A2expression was not detected. Among conjugative enzymes, wefound increased mRNA levels of COMT (p < 0.0001) andunchanged mRNA levels of SULT1A1 and NQO2, while all theothers gene levels were significantlly decreased: UGT2B7, NQO1,GSTP1, SULT2B1 (p < 0.0001), and SULT1E1 (p = 0.0311). OurqPCR data thus suggest increased phase I and decreased phaseII metabolism of estrogens that leads to exessive ROS formation,which may activate cytokines that control the implantation and thegrowth of endometrial cells outside the uterus and thus contributeto the development of ovarian endometriosis.

P030

Evaluation of Various Concentration Methods forDetecting Enterovirus in Water

Bing-Mu Hsu

National Chung Cheng University, Taiwan, Republic of China

Enteroviruses generally spread through the fecal-oral route, butthe importance of viral transmission by water is probablyunderestimated. A reliable, sensitive, and practical method fordetecting small concentrations of enteroviruses is needed. Theobjective of this paper is to evaluate series of procedures toconcentrate enteroviruses by adsorption to and elution fromvarious charged membranes for various types of water. Therelative recovery efficiencies of enterovirus in treated watersamples were also evaluated by Real-Time PCR. The optimumprocedures designed for enteroviruses detection of theenvironmental water samples were done in this study. Two stageprocesses, prefiltration and adsorption, were used for separatingenteroviruses from large volume of water sample. The watersamples passed through prefilter apparatus were collected, andthen were filtered through the evaluated membranes foradsorption. The types of adsorbing disc filters evaluated in thisstudy includes positive charged membrane (6408502 1MDS, 47-mm diameter, CUNO Inc., Meriden, Conn. U.S.A.), negativecharged membrane (NM04701 020SP, 47-mm diameter, CUNOInc., Meriden, Conn. U.S.A.), and nitrocellulose membrane (HAseries, 47-mm diameter, 0.45-um pore size, Millipore, Ireland).The relative recovery efficiencies of various concentrationmethods for enteroviruses were analyzed by Real-Time PCR and

shown in the Table 1. The optimum procedure is followed as thatthe water sample was prefiltered first. The prefiltered water wasadjusted pHs and electrolyte, then passed through nictrocellulosemembrane. Aliquot of H2SO4 was passed through the membraneto rinse out the cation, and then NaOH was poured on themembrane and the filtrate was recovered in a tube containingH2SO4 and TE buffer for neutralization. All of the eluate wasconcentrated with an ultrafiltration system. The contents ofenvironmental inhibitors after virus concentration are suspected tohave some inhibitory effect on viruses PCR detection. It should beconsidered while detecting enteroviruses in environmentalsamples.

P031

Event-specific detection of genetically modified rice(Kefeng 6) by means of quantitative real-time PCR

Patrick Guertler, Ingrid Huber, Sven Pecoraro, Ulrich Busch

Bavarian Health and Food Safety Authority, Germany

Rice is one of the most important food crops in the world,especially in Asian countries. However, heavy losses in rice yield(24%-41% per year) are caused by rice insect pests. To addressthis issue, several varieties of genetically modified (gm) rice havebeen developed and released for field trials in China. One cropunder study is the insect resistant rice called “Kefeng 6”. Thistransgenic rice line has been altered by insertion of genesencoding for Cry1Ac and CpTi, both proteins responsible for anincrease insect resistance. The hygromycin resistance gene (hpt)has been inserted as a selective marker. Despite the fact that gmrice has not been approved for commercial cultivation in China (orelsewhere in the world), there were reports on illegal planting andtrade of gm rice in a central province of China. By way of theglobal trade, Kefeng 6 rice was found in food samples in theNetherlands in 2010 (rapid alert system for food and feed, RASFFNo. 2010.0336). In the European Union (EU), Kefeng 6 is notauthorized for import, processing or cultivation and materialcontaining Kefeng 6 is therefore not permitted at any level. Inorder to monitor the potential presence of Kefeng 6 rice inproducts imported into the EU, a sensitive and specific event-specific detection method needs to be on hand to clearlydistinguish between different gm rice lines. Therefore, it was theaim of this study to develop an event-specific detection method forthe gm rice Kefeng 6 by using quantitative real-time PCR (qPCR)and to validate this method according to the guidelines of GMOtesting. Afterwards, this method was used to monitor several riceproducts on a small scale level. Based on the flanking sequenceof the DNA insert, primers and a hydrolysis probe were designedand used in a qPCR assay applying the ABI TaqMan technology.The assay was validated as outlined in the “Minimum performancerequirements for analytical methods for GMO testing” and ispresented as outlined in the MIQE guidelines. The validationprocess revealed a limit of detection of 5 copies of the transgenewith confidence intervals (95%) between 0.07 and 0.52, a meanPCR efficiency of 105 % and a mean R2 of 0.99. The specificitywas determined by analysis of different gm and non-gm crops byusing this assay. No positive amplification signal was observed forany of the analyzed crops. Furthermore, the assay was tested byanalysis of rice samples obtained on the local market. Only onesample, that has been previously tested positive by means of aconstruct-specific detection assay, was again tested positive byusing this assay. This underlines the suitability of this assay for themonitoring of food samples for the presence of Kefeng 6 rice.

P032

Examining resistance gene expression using reversetranscriptase qPCR

Tine Yding Wolff1, Jan Lorenzen1, Trine Rolighed Thomsen1,2,Anne Karin Ildor Rasmussen3, Henrik Stender3, HelleStendahl Andersen1, Masumeh Chavoshi1

1The Danish Technological Institute, Denmark; 2AalborgUniversity, Denmark; 3AdvanDx, Denmark

Objective: The aim of the study was to examine the expression ofthe mecA gene in clinical methicillin-resistant Staphylococcusaureus (MRSA) isolates upon induction with cefoxitin usingreverse transcriptase qPCR (RT-qPCR). Methods: 18 S. aureusstrains (16 MRSA representing staphylococcal cassettechromosome mec (SCCmec) types I-V and 2 MSSA) were grown


with shake at 35°C for 40 min with and without cefoxitin.RNAprotect Bacteria Reagent (Qiagen) was added to 1 mLsuspensions of each culture and the cells were disrupted byenzymatic (lysostaphin) and mechanical (bead beating) lysis. TotalRNA was extracted using the RNeasy PLUS mini kit (Qiagen)including the optional DNAse treatment step. cDNA wassynthesized from 100 ng RNA using the SuperScriptTM III FirstStrand Synthesis System with random hexamers (Invitrogen). ThecDNA was template in qPCR reactions targeting the mecA and the16S rRNA genes respectively. On basis of the qPCR data thebaselines for cefoxitin induced and uninduced cells and the meanfold changes (MFC) in mecA expression were calculated asdescribed by Shang et al. (2010). The procedure was carried outthree times for each strain and to evaluate the effect of inductionon mecA expression uninduced and induced baselines werecompared by paired Students’ t-tests. The minimum inhibitoryconcentration (MIC) of cefoxitin was determined for each isolateusing E-test. Results: Significant changes in the expression ofmecA upon cefoxitin induction (P<0.05) were observed for sevenof the MRSA isolates representing three different SCCmec types(type II (n=2), type IV (n=3) and type V (n=2)). Throughout thestudy no correlation between the SCCmec type and the level ofmecA expression was observed. The cefoxitin MICs of the MRSAisolates ranged from 12 to 256 µg/mL and there was a tendencyfor strains with a low cefoxitin MIC to have a low mecA expressionas well. Conclusion: It was possible to apply RT-qPCR for thedetection of changes in the mecA expression upon cefoxitininduction. Significant changes (P<0.05) were observed for sevenof the examined MRSA isolates. There was no correlationbetween the SCCmec type and the induced baselines, but itseemed that isolates with a low cefoxitin MIC also had a lowexpression level of mecA.

Shang et al, Antimicrob Agents Chemother 2010, p. 956-59.

P033

Experience with Plant RNA Isolation Aid for the totalRNA isolation from plants

Irena Mavric Plesko1, Mojca Virscek Marn1, Natasa Toplak2,Minka Kovac2

1Kmetijski institut Slovenije, Slovenia; 2Omega d.o.o., Slovenia

Plant tissue is known as a difficult tissue for RNA extraction sinceit can contain big amounts of secondary metabolites, such aspolysaccharides and phenolics. Some of these compounds areknown PCR inhibitors. We investigated the influence of Plant RNAIsolation Aid (Life Technologies) on quality and quantity ofextracted total RNA (totRNA) from different plant tissue. TotRNAwas extracted on MagMAX™ Express Magnetic ParticleProcessor using MagMAX™-96 Total RNA Isolation Kit (LifeTechnologies) with and without addition of Plant RNA IsolationAid. The analyzed plant tissues included raspberry leaves, in vitropotato plantlets, potato tubers, grapevine leaves and tomatoleaves. Extracted totRNA was reverse transcribed and its quantitywas checked using 18S rRNA qPCR assay. The results of qPCRshow no differences when Plant RNA Isolation Aid was used fortotRNA extraction from potato and tomato. With raspberry andgrapevine tissue the differences in Ct values were between 3 and19. The results of our study show that Plant RNA isolation Aid canimprove RNA extraction from certain plant species.

P034

Expression of Laccase genes in Schizophyllumcommune

Soumya Madhavan, Katrin Krause, Erika Kothe

Friedrich Schiller University, Institute of Microbiology, MicrobialPhytopathology, D-07743 Jena, Germany

Schizophyllum commune, a saprophytic white rot fungus, isinvolved in the degradation of complex organic moleculesincluding lignin. Previous reports say that this fungus can degraderefractory organic matter from black slate with the help of differentexoenzymes which perform radical reactions to oxidize organicmatter. The genome of S. commune reveals 16 FOLymes (FungalOxidative Lignin Enzymes), which are potentially involved in lignindegragation. These genes include three lignin oxidases (LO) and13 lignin degrading auxiliary enzymes (LDA) genes. In this study,relative expression of the laccase and laccase-like genes (LO

family) in S. commune areinvestigated using quantitative real timePCR (RT-PCR). Two laccases and four laccase-like genes wereselected for expression studies based on microarray datacomparing S. commune grown on a black slate surface and grownon complex yeast medium. Also, the involvement of laccases inhyphal morphogenesis, especially during fruitbody formation isinvestigated by analyzing the relative expression of the genes ofinterest in hyphae and fruitbody tissue. Preliminary results revealthat laccase expression is upregulated in the cultures with blackslate powder. This shows that laccase and laccase-like genes areinfluenced by the presence of black slate in minimal mediumwhich is to be expected if laccases are involved in the degradationof stone material.

P035

Expression of local luteotropic factors during inducedluteolysis in the bovine corpus luteum

Dieter Schams1, Heinrich Meyer1, Bajram Berisha1,2

1Physiology Weihenstephan, Technische Universität München,Germany; 2Faculty of Agriculture and Veterinary, University ofPrishtina, Kosovo

The essential role of endometrial prostaglandin F2 alpha (PTGF)for induction of the corpus luteum (CL) regression is welldocumented in the cow. However, the acute effects of PTGF onknown local luteotropic factors: oxytocin (OXT) and its receptor(OXTR), insulin-like growth factor 1 (IGF1), and progesterone(PG) and its receptor (PGR), were not thoroughly studied in detail.The aim of this study was therefore to evaluate the tissueconcentration of these factors during PTGF induced luteolysis. Inaddition the mRNA expression of PGR, OXTR, IGF1, insulin-likegrowth factor binding protein 1 (IGFBP1), was determined at welldefined times after PTGF treatment. Cows (n=5 per group) in themid-luteal phase (Days 8–12, control group) were injected with thePTGF analog (cloprostenol), and CL were collected bytransvaginal ovariectomy at 0.5, 2, 4, 12, 24, 48, and 64 h afterinjection. The mRNA expression was analyzed by quantitativereal-time RT-PCR, and the protein concentration in tissue wasevaluated by enzyme immunoassay or radioimmunoassay. PGconcentrations, as well as mRNA expression of PGR, in CL tissuewere significantly down regulated by 12 h after PTGF. Tissue OXTpeptide and OXTR mRNA decreased significantly after 2 h,followed by a continuous decrease of OXT mRNA. IGF1 proteinalready decreased after 0.5 h. By contrast, the IGFBP1 mRNAwas up-regulated significantly after 2 h to a high plateau. Theacute decrease of local luteotropic activity may be a keycomponent in the cascade of events leading to functionalluteolysis in the cow.

P036

Expresssion of Adrenomedullin signalling factors inbovine corpus luteum during oestrous cycle,pregnancy and induced luteolysis

Bajram Berisha1,2, Stefanie Schilffarth1, Mihir Sarkar1, DieterSchams1

1Physiology Weihenstephan, Technische Universität München,Germany; 2Faculty of Agriculture and Veterinary, University ofPrishtina, Kosovo

Investigations on "nonclassic" regulators of angiogenesis couldopen new perspectives in understanding angiogenesis underphysiological and pathological conditions. A pro-angiogenic effecthas been demonstrated for adrenomedullin (AM), it´s co-factoractivity-modifying protein 2 (RAMP-2) and the Calcitonin receptor-like Receptor (CALCLR). This signalling pathway is besidesassociated with lymphangiogenesis, that (in a physiologically way)is still only poorly understood in ovary and its structures. The aimof this study was therefore to evaluate mRNA expression of AM,RAMP-2 and CALCLR in bovine corpora lutea (CL) duringdifferent physiological stages. Experiment 1: CL were assigneddays 1-2, 3-4, 5-7, 8-12, 13-16, >18 (after regression) of oestrouscycle and of gravidity (month <3, 3-5, 6-7 and >8). Experiment 2:Induced luteolysis. Cows on days 8-12 were injected with aPGF2α analogue and CL were collected by transvaginalovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after PGF2αinjection. Tissue levels of mRNA were characterized by real-timeRT-PCR. All 3 factors were clearly expressed. AM and CALCRL


showed significant changes in both experiments, RAMP-2 duringinduced luteolysis. For AM highest levels could be observed at thebeginning and the end of the luteal phase, CALCRL remained atcomparable levels. Both factors showed their highest expressionat the end of pregnancy. After induced luteolysis the expression ofall factors started to decline (significantly during structuralluteolysis). In conclusion, our results could lead to the assumptionthat factors investigated may be involved in mechanismsregulating CL formation, function and regression – with a specialemphasis during the final months of pregnancy.

P037

Gene Expression of Selected WRKY TranscriptionFactors in Arabidopsis thaliana Cell Cultures during aParabolic Flight

Anne Hennig, Maren Neef, Niklas Hausmann, Rüdiger Hampp

University of Tuebingen, Physiological Ecology of Plants,Germany

Plants use gravity as a guide for growth and development. Recentstudies could demonstrate that plant cells which are not part ofspecialized tissues such as the root columella cells are also ableto sense gravitational forces. Therefore we used undifferentiated,homogeneous cell cultures of Arabidopsis thaliana (cv. Columbia)(A.t.) in order to identify early alterations in gene expression as aresponse to altered gravitational fields. As experimental systemwe used parabolic flights (A300, Novespace, France). A singleparabola consists of following phases: 1g, 1.8g for 20 sec,microgravity (µg) for 22 sec, 1.8g for 20 sec, 1g. Up to 30parabolas are exercised within one flight. By the use ofmicroarrays (ATH1, Affymetrix) we found several hundredtranscripts to be altered in amount during the respective gravitysegments. For this study, we specifically looked for genes at thebeginning of signal transduction chains, such as transcriptionfactors (TFs). TFs are small proteins regulating gene expressionof their target genes by binding to specific promoter sequences.For quantification via qRT-PCR, six WRKY genes were selected.WRKYs are known to be involved in various physiologicalprocesses such as senescence or pathogen defense. The dataindicated a transient gene expression profile, while most of theanalyzed WRKYs were significantly down-regulated after themicrogravity-phase of the parabola.

P038

Gene expression studies on soil fertility and nutritionalstatus in grapevine

Nadia Bertazzon1, Piergiorgio Stevanato2, Alessandro Zonin2,Giuseppe Concheri2, Massimo Saccomani2, Elisa Angelini1

1C.R.A. Centro di Ricerca per la Viticoltura, 31015, CONEGLIANO(TV), Italy; 2Università degli Studi di Padova, Dipartimento diBiotecnologie Agrarie, 35020, LEGNARO (PD), Italy

Grapevine is a major fruit crop worldwide that is susceptible toseveral environmental stresses like diseases and water-nutritionaldeficiency. Its management involves many agronomic practicesand phytochemicals to prevent significant reductions in quality andquantity of production caused by these stresses. There is anurgent need for developing alternative sustainable strategies forgrapevine protection. This requires innovative and interdisciplinaryapproaches for a better understanding of grapevine responses toenvironmental stresses. The aim of this study was to evaluate therelationships among grapevine productivity, main soil chemicalproperties and expression of genes that could be involved indefence mechanism of grapevine to edaphic stresses. Theresearch was conducted in two vineyards characterized by highand low productivity, both cultivated with cv. Garganega on 1103Paulsen and located in the North-East of Italy. The evaluation ofthe soil properties revealed large and significant differences(p<0.001) between the two vineyards. The soil of vineyardcharacterized by high productivity showed neutral pH, good supplyof organic matter and appropriate C/N ratio (8:1). On the contrary,the soil of less productive vineyard was characterized by acid pH,scarce organic matter content, suboptimal C/N ratio and leavecontent of nitrogen and sulphur. Four selected defence-relatedgenes were analysed by qPCR starting from total RNA ofgrapevine leaves collected on August 2010 from the twovineyards. Two genes, phenylalanine ammonia lyase (PAL) and

stilbene synthase 1 (VST1), encode key enzymes of thephenylpropanoid pathway which leads to the production of variousdefence-related compounds, like resveratrol. Another geneencodes the enzyme sucrose synthase (Susy) which is involved inthe metabolism of sucrose. The last gene encodes for a WRKYtranscription factor, implicated in the regulation of transcriptionalreprogramming associated with plant immune responses. A set offive commonly used Vitis vinifera reference genes, encoding actin(ACT), cytochrome oxidase (COX), pyruvate dacarboxilase (PDC),glyceraldehyde-3-phosphate dehydrogenase (GPDH) and 26Sribosomal RNA (26S), were used to normalize expression data.Two genes, GAPDH and ACT, were selected among them for thenormalization process, as they showed stable expression in all ourexperimental conditions. All the target genes analyzed showedsignificant higher relative expression values (p<0.001) in plantsfrom low-productive vineyard with respect to those from high-productive vineyard. These results showed that deficiencies in soilfertility and nutritional state of grapevine lead to the induction ofthe selected defence-related genes which could be involved inphenotypic adaptation of vine plants to edaphic stress. FurtherqPCR experiments will be carried out for the validation of thesegene expression patterns on nitrogen and sulphur-starved plantsgrown under controlled conditions.

P039

H275Y screening and quantification using isolatedunlabeled probe

Hong Kai Lee1, Chun Kiat Lee1, Julian Wei-Tze Tang1, EvelynSiew-Chuan Koay1,2

1Molecular Diagnosis Centre, Department of Laboratory Medicine,National University Hospital, Singapore; 2Department ofPathology, National University of Singapore, Singapore

Objectives: An increasing number of oseltamivir-resistanceassociated mutations (H275Y in the neuraminidase NA gene) arebeing reported in H1N1/2009 viruses isolated from patients treatedwith oseltamivir, particularly in the immunocompromised.1 It isimportant to identify the presence of these drug resistance-associated mutations to optimize the care for such patients.Methodology: A rapid and cost-effective High Resolution Melting(HRM) PCR assay was developed for this purpose using anunlabeled probe to screen for the H275Y mutation among patientstreated with oseltamivir, especially the highly susceptibleimmunocompromised patients. Isolated probe PCR (IP-PCR)2 wasperformed using Superscript III Platinum One-step qRT-PCR kit(Invitrogen) on either the LightScanner LS32 (Idaho) or theLightCycler LC v2.0 (Roche) system, both of which utilize theuniquely designed LC glass capillaries as reaction receptacles. Toinitiate the IP-PCR, 2 μL of extracted viral RNA was added to 6 μLof reaction mix containing optimized amounts of forward andreverse primers. A calculated excess amount of forward primerand unlabelled probe mix (2 μL) was then aliquotted into the upperwell of the capillary tube, without being spun down with thereaction mix. Upon completion of the IP-PCR, the 8 μL reactionmix was mixed with the pre-deposited primer/probe mix byinversion and reverse-spinning, followed by an ”extended” 15-cycle asymmetric amplification reaction and continuous HRM dataacquisition. The in vitro synthesized RNA transcripts for bothwildtype and mutant were diluted using pooled negative RNAextracts and used as the standard calibrator. The lower limit ofdetection (LLOD) and lower limit of quantification (LLOQ) for themutant strain were determined. To estimate the proportion ofH275Y mutant population in a mutant/wildtype mixture, a series of0-100% mixed-mutant/wildtype standards were tested, at 10%incremental intervals and 105 RNA copies/reaction. Results: Themelt peaks for the unlabeled probe with a mismatched base C(wildtype) and that with a perfectly matched base T (mutant) wereclearly discernable at 65.5°C and 69.0°C, respectively. The assayreproducibly amplified concentrations of 20 to 2x107

RNAcopies/reaction, thus giving an LLOD of 20 copies/reaction.The LLOQ, defined as the lowest concentration whereby thedifference of 3.32 Ct value per log is maintained, was 200copies/reaction. Good resolution of the melt peaks was obtainedfor the whole series of 0-100% mixed-mutant/wildtype standards.Conclusion: This assay is able to provide easily discernableresults for qualitative and quantitative detection of WT(H275)/mutant (Y275) mixes. The preliminary results suggest thatthis assay and platform may accurately identify H275Y populationsdown to 10% of the viral population, though further validation onclinical specimens is required.


1. WHO, Wkly Epidemiol Rec 2010;85:37-40

2. MD Poulsen, CT Wittwer. Biotechniques 2007;43:87-91

P040

Improvement of phytoplasma diagnostics in grapevineand fruit trees: from grinding to real time PCR analysis

Matevž Rupar, Nina Prezelj, Nataša Mehle, Petra Nikolić, MajaRavnikar, Marina Dermastia

National Institute of Biology, Slovenia

Phytoplasmas are plant pathogenic bacteria without cell wall andmay cause severe damage to their plant hosts. Examples of suchpathogens are Flavescence doreé (FD) and Bois noir(BN)phytoplasma causing grapevine yellows and ‘CandidatusPhytoplasma mali’ (AP), ‘Ca. P. prunorum’ (ESFY) and ‘Ca. P.pyri’ (PD) from apple proliferation group phytoplasmas on fruittrees. All of them can cause great crop losses and economicaldamage if not detected soon enough and properly managed.Because phytoplasmas cannot be grown in vitro, their diagnosticsdepends only on molecular and serological methods. The mostcommonly applied assays involve laborious and time consumingsteps of DNA extraction followed by the conventional PCR andseveral PCR- RFLP analyses with agarose or polyacril amid gelelectrophoresis. Through recent years our laboratory developedand improved methods for fast, Real time PCR based diagnosticsof phytoplasma in grapevine and fruit trees with higher sensitivity,specificity and fewer possibilities for contaminations. Wecompared the homogenization of plant tissues using mortar andpistil in liquid nitrogen with a Fast Prep® homogenizer andadditionally compared both methods using real-time PCRdescribed below. For a DNA extraction CTAB method andKingFisher® automatic extraction procedure based on magneticbeads (Pirc et al., 2009) were compared with pipetting of a largeset of samples using an automatic pipetting workstation. ThePCR-PFLP analyses were compared with a real-time PCR assay,using different primers and probes. We use primers and probes for18S rRNA gene (Applied Biosystems, USA) as an internal controlof DNA isolation, primers and probes for universal phytoplasma(Hren et al., 2007) detection phytoplasma and from appleproliferation group (AP, PD, ESFY) (Nikolić et al., 2010). With anew procedure, which combines a homogenization with a FastPrep® homogenizer, KingFisher® automatic extraction procedurebased on magnetic beads, pipetting of a large set of samples byan automatic pipetting workstation and real-time PCR assays weincreased the number of processed samples in time, diminishedthe possibilities of contaminations, improved the sensitivity andspecificity of detection and shortened the time for the diagnosis forapproximately three times. Therefore, new protocols combinedenables high throughput routine phytoplasma diagnostics.

Hren M., Boben J., Rotter A., Kralj P., Gruden K., Ravnikar M. (2007). PlantPathology 56 (5), 785-796.

Nikolić P., Mehle N., Gruden K., Ravnikar M., and Dermastia M. (2010).Molecular and Cellular Probes 24: 303-9.

Pirc M., Ravnikar M., Tomlinson J., Dreo T. (2009). Plant Pathology, 58 (5):872-881.

P041

Influence of different one-step reverse transcriptionreal-time PCR reagents for no-doubt detection ofnorovirus genogroup II

Marko Kolenc1, Nataša Toplak2, Minka Kovač2, Andrej Steyer1,Mateja Poljšak Prijatelj1

1University of Ljubljana, Faculty of Medicine, Institute ofMicrobiology and Immunology, Slovenia; 2Omega d.o.o., Slovenia

Human noroviruses, members of Caliciviridae family are one ofthe main cause of acute non-bacterial gastroenteritis in humans ofall ages. They appear in both, epidemic and sporadic cases.Noroviruses can not be propagated in cell cultures. Thus real-timePCR molecular methods for caliciviral detection, which representthe basis of molecular epidemiology, have been introduced [1,2,3].The aim of the study was to compare two different real-time PCRreagents for detection of norovirus strains belonging to genogroupII. The linear range of detection, sensitivity, efficiency ofamplification, comparison of detected fluorescence using twodifferent one-step reverse transcription real-time PCR reagentswere established. The results show that the use of a TaqMan Fast

Virus 1-Step Master Mix (Applied Biosystems by LifeTechnologies) offers fast, sensitive and reliable (robust) detectionof norovirus genogroup II in comparision to the reagent fromanother supplier.

1] Dingle, K.E., Lambden, P.R., Caul, E.O. and Clarke, I.N. 1995. Humanenteric Caliciviridae: the complete genome sequence and expression ofvirus-like particles from a genetic group II small round structured virus. JGen Virol 76: 2349-2355.

2] Hoehne, M., Schreier, E. 2006. Detection of Norovirus genogroup I and IIby multiplex real-time RT- PCR using a 3'-minor groove binder-DNA probe.BMC Infectious Diseases 2006,

http://www.biomedcentral.com/1471-2334/6/69

3] Kageyama, T., Kojima, S., Shinohara, M., Uchida, K., Fukushi, S.,Hoshino, F.B., Takeda, N., Katayama, K. 2003. Broadly reactive and highlysensitive assay for Norwalk-like viruses based on real-time quantitativereverse transcription-PCR. J Clin Microbiol, 41 (4): 1548-1557.

P042

Interest of Real-Time PCR for the Diagnosis of InvasiveAspergillosis

Inès Hadrich1, Fattouma Makni1, Sourour Neji1, FatmaCheikhrouhou1, Hayet Sellami1, Stéphane Ranque2, Ali Ayadi1

1Habib Bourguiba Hospital: Sfax - Tunisia, Tunisia; 2Laboratoirede Parasitologie-Mycologie, Université de la Méditerranée, AP-HMTimone, Marseille France

Background. Improving our ability to detect Aspergillus DNA willcontribute to the diagnosis of invasive aspergillosis (IA). This studyaimed at comparing a real-time PCR assay and a PCR-ELISAassay in both serum and bronchoalveolar lavage (BAL) samplesforthe diagnosis of IA in patients with hematological malignancies.Patients and methods. 163 neutropenic patients at risk of IA in thehematology wards of the Sfax University Hospital, wereprospectively studied. BAL samples, if available, and serum weretaken twice weekly. The morphological identification of Aspergillusisolates collected from BAL samples was verified by internaltranscribed spacer 1 (ITS1), 5.8S, and ITS2 region rRNAsequence analysis. Using a nested case-control design, both PCRassays were performed on proven and probable IA cases andmatched control samples. Results. One proven, 31 probable, and15 possible cases of IA were diagnosed on the basis ofEORTC/MSG criteria. The ITS sequencing proved that all isolatescollected from BAL were Aspergillus flavus. Real-time PCR, PCR-ELISA, and galactomannan antigen (GMA) assays performed on459 serum samples found 93.8%, 96.9%, and 100% sensitivity,respectively. Specificity was 100%, 100%, and 91.5%,respectively. In 42 BAL samples, sensitivity was 64.3%, 71.4%,and 85.7%, and specificity was 96.4%, 96.4%, and 92.9%,respectively. Conclusions. While slightly less sensitive, the realtime-PCR assay was highly specific and considerably faster andmore workable than PCR-ELISA. Combining real-time PCR andGMA detection in both serum and BAL samples enhances routinelaboratory IA diagnosis.

P043

Is the JAK2 Exon 14 Deletion a Physiological SpliceVariant?

Paolo Catarsi, Vittorio Rosti, Laura Villani, Valentina Poletto,Elisa Bonetti, Bergamaschi Gaetano, Barosi Giovanni

Unit of Clinical Epidemiology and Center for the Study ofMyelofibrosis, IRCCS Policlinico San Matteo Foundation, Italy

In recent years the diagnosis and treatment of patients withmyeloproliferative disorders has focused on the JAK2 gene. TheV617F mutation changes the pseudokinase domain JH2 anddetermines the constitutive activation of the protein. In a recentpublication, Ma and colleagues (Ma et al.. PLoS ONE (2010) vol. 5(8)) identified an alternative splicing of JAK2 transcript that leadsto the deletion of the entire exon 14 (88bp). This mRNA has a stopcodon in exon 15 and is translated into a truncated protein thathas a deletion within the JH2 pseudokinase domain and completedeletion of the kinase domain (JH1). In order to identify andquantify the presence of this splice variant, the RNA extractedfrom plasma, was reverse transcribed and amplified with primersdesigned in exons 13 and 15 and analyzed using fluorescentfragment length analysis. The alternative transcript was onlydetected in patients with myeloproliferative neoplasms (MPNs)(58% of patients positive for the V617F mutation and 33%


negative) in proportions ranging from 2% to 26% compared to thenormal transcript. We investigated this alternative splicing form inRNA extracted from both granulocytes and total leucocytes of 6MPNs patients (3 positive for the V617F mutation and 3 negative)and 5 healthy controls. The amplification products (i.e. 273-bp forthe wild type and 185-bp for the splice variant) were obtainedusing the same pair of primers. The products were separated byconventional gel electrophoresis. The alternative spliced productwas present in all patients and all healthy individuals. Wehypothesized that the difference between patients and controlsgroups observed in the cited work is due to a pathologicalincrease in the levels of a constitutive variant. To test thishypothesis, we are developing a qPCR assay to determine theamount of JAK2 splice variant relative to the normal transcript.

P044

Molecular Detection of Tranzschelia discolor usingqPCR

Paolo Catarsi1,3, Cristina Borghi1, Marina Laura1, AriannaCassetti1, Giacomo Morreale2, Andrea Allavena1

1CRA-FSO, Research Unit for Floriculture and OrnamentalSpecies, Corso Inglesi 508, 18038 Sanremo, Italy; 2CRA – VIT,Centre for Research in. Viticulture, Viale XXVIII Aprile 26, 31015,Conegliano, Italy; 3Current address: Lab of Clinical Epidemiology,IRCCS Policlinico San Matteo Foundation, Piazzale Golgi, 27100Pavia, Italy

Prunus/Anemone rust is caused by Tranzschelia discolor, that hasbecome aggressive in Anemone coronaria cultivation in recentyears. Reliable detection of plant pathogens in propagationmaterial crops and ornamental plants is matter of great economicinterests because of farmer yield losses. Furthermore the supplyof of infected seed can lead to protracted litigation. Aim of ourstudy was to develop a qPCR technique useful to detectTranzschelia discolor in both seed derived rhizomes (i.e. thepropagation material of Anemone) and in epigeal tissues of theplants. This technique can be useful for both its phytosanitaryapplication (i.e. the detection of the pathogen in plant nurseryfacilities) and its usage as a powerful method to detect thepresence of mycelium in various tissues of the Anemone plantduring fungus migration. qPCR primers were designed on a DNAsegment coding for a ribosomal RNA. The qPCR reaction allowedus to efficiently detect the fungus in seed derived rhizomes. Thisfinding is in agreement with the hypothesis that infectedpropagation material accounts for yield losses in the field. qPCRanalysis confirmed the migration of the fungus in epigeal tissues ofthe plant. Furthermore, qPCR data were used to set up a LAMPbased procedure, which might allow pathogen on-site detection.

P045

Scaling Semiconductor Sequencing from Microbial toHuman Genomes on a Single Platform

Alain Rico1, Jonathan M. Rothberg2

1Life Technologies, France; 2Ion Torrent, USA

Ion Torrent has invented the first device—a new semiconductorchip—capable of directly translating chemical signals into digitalinformation. The first application of this technology is sequencingDNA. The device leverages decades of semiconductor technologyadvances and in just a few years has brought the entire design,fabrication and supply chain infrastructure of that industry - atrillion dollar investment—to bear on the challenge of sequencing.The result is Ion semiconductor sequencing, the first commercialsequencing technology that does not use light, and as a resultdelivers unprecedented speed, scalability and low cost. All ofthese benefits are a result of applying a technology that ismassively scalable, as proven by Moore’s Law, to a task that hastraditionally used optics-based solutions. Optics-basedsequencing works in a linear fashion: Increasing capacity requiresincreasing the number of signals that must be read, which in turnresults in longer runtimes, higher capital costs and ever moresophisticated optics.

P046

Multiplex Real-Time PCR for Detection of Shiga toxinand Intimin genes of pathogenic Escherichia coli

Melanie Pavlovic, Regina Konrad, Sabine Wolf, MarionLindermayer, Jasmin Fräßdorf, Ulrich Busch, Ingrid Huber

Bavarian Health and Food Safety Authority, Germany

Some E. coli strains harbour specific virulence factors whichclassify them as pathogens. In human they mainly causeinfections of the gastrointestinal and urinary tracts and are amongthe most frequent bacterial causes of diarrhea. Especially in youngchildren, infections with Shiga toxin producing enterohaemorrhagicE. coli (EHEC) can lead to the life threatening hemolytic uremicsyndrome (HUS). EHEC and enteropathogenic E. coli (EPEC)strains can induce attaching and effacing lesions. They harbor thelocus of enterocyte effacement (LEE) pathogenicity island with theintimin gene eae. Intimin mediates the intimate attachment ofbacteria to epithelial cells. The diagnosis of EHEC strains in stooland food is based on detection of Shiga toxin genes and of EPECstrains on the eae gene, respectively. We developed a multiplexreal-time PCR assay for simultaneous detection of intimin genesand all so far known variants of Shiga toxins 1 and 2 (stx1 andstx2) for diagnostic samples on a Lightcycler® 480 machine withhydrolysis probes (Pavlovic et al., 2010). An internal amplificationcontrol was included. The multiplex PCR was tested on 30 E. colireference strains and 174 well characterized isolates from ourroutine diagnostic. 68 strains representing other gastrointestinalpathogens, normal gastrointestinal flora, or closely related bacteriagave no signal in the multiplex PCR. The detection limits were fivegenome equivalents for stx2 and 50 genome equivalents for eaeand stx1. Before implementing the multiplex PCR in the routinediagnostic, we compared a set of samples examined with thenewly developed method and the currently used real-time PCR.Overall 95% of the results were identical for the stx1 and stx2genes (n = 182 each) and 91% (n = 54) for the eae gene. In factwe detected more stx and eae genes in our samples with the newmultiplex PCR because all variants of the genes are included.Additional samples are currently tested.

Pavlovic M, Huber I, Skala H, Konrad R, Schmidt H, Sing A, Busch U.Development of a multiplex real-time polymerase chain reaction forsimultaneous detection of enterohemorrhagic Escherichia coli andenteropathogenic Escherichia coli strains. Foodborne Pathog Dis. 2010Jul;7(7):801-8.

New qPCR Applications:Method Optimisation & Standardisation

P047

Hot Start dNTPs – Novel Chemistries for Use inAdvanced PCR Applications

Natasha Paul

TriLink BioTechnologies, Inc., United States of America

PCR is a widely used scientific tool whose specificity can beincreased by the use of Hot Start technologies. Although manyHot Start technologies exist, recently developed CleanAmp™dNTPs are a distinct approach that employs modified nucleosidetriphosphates with a thermolabile protecting group at the 3'-hydroxyl. The presence of the protecting group blocks lowtemperature primer extension, which can often be a significantproblem in PCR. At higher temperatures, the protecting group isreleased to allow for incorporation by the DNA polymerase andmore specific amplification of the intended target. These modifieddNTPs provide comparable performance to other Hot Starttechnologies and can be used with thermostable DNApolymerases to turn a reaction into a Hot Start version. Thisthermolabile chemistry can be applied to dNTP analogs such asdUTP, which is used in UNG decontamination methods, and 7-deaza-dGTP, which is used to amplify difficult GC-rich targets. Inaddition, further studies have led to the development of 3'-protecting groups that deprotect more quickly than the current 3'-modification group, allowing these modified dNTPs to be used infast PCR. With the evolving chemistry of the hot start dNTPs, the


areas of application benefiting from the versatility and flexibility ofthis technology continue to grow.

P048

Lyophilized Mix of Enzyme, Probes, and Primers: Asimple and highly sensitive solution for qPCRapplications.

Nanette Umblas, Chieh-Yuan Li, Lin Lin Huang, Kathy Lee,David Keys, Kate Hames, Madhu Augustine, StephenHendricks


qPCR reactions require a number of components includingdNTPs, salts, buffers, enzyme, qPCR assay primers and probes,and sample. Numerous pipetting steps may introduce errors andare not amenable for high throughput applications. A number oftime consuming pipeting steps can be eliminated when you usepre-formulated primer/probe mixes such as Applied Biosystems’TaqMan® Gene Expression Assays, with optimized enzymemixes. However, a single pipetting step workflow from sample toqPCR reaction is highly desirable for new applications, especiallyin clinical research and molecular diagnostics, where minimalhandling is often required. Thus, a solution that provides ease ofuse, without compromising precision and accuracy is highlyadvantageous. To meet this need we have developed a qPCRsolution that centers on lyophilization of probe, primers andenzyme mix. Lyophilization, also known as freeze drying, is awidely used technique in food and pharmaceutical industries forroom temperature storage and transportation of compounds. Wehave successfully applied this technique to qPCR such that probeand primers for any gene can be combined with enzyme into a mixthat is stable at room temperature. With the new lyophilizedformat, end users only need to add their sample solution into thereaction tube (8-well tube strips or 96-well plates) where thelyophilized assay and enzyme mix has been pre-dispensed.Besides providing convenience, cost savings and minimalpipetting error, this format allows increased sample volume, thus,potentially doubling the sensitivity of detection compared to wetassays. Here we present the challenges of applying lyophilizationin qPCR, lyophilized assay performance data when compared towet assays (R^2 = 0.98; PCR efficiency ~100%), and preliminarystability data when stored under different conditions.

P049

Extraction protocol to increase the yield of circulatingfetal DNA in maternal plasma

Lai-on Chu, C.Y. Rachel Yeung, K.F. Joseph Chow

DiagCor Bioscience Inc. Ltd., Hong Kong S.A.R. - China

Background: Compare to the current invasive prenatal diagnosismethods,non-invasive approach is an ideal way to performdiagnostic test in much earlier stage of pregnancy while reducingthe chance of miscarriage minimum. However, the nature of lowconcentration of cell free fetal DNA (cffDNA) in maternalcirculation is the major technical challenge to achieve effectivenon-invasive prenatal diagnosis. We present here a samplecollection and extraction workflow generating substantial increasein extraction yield and total amount of fetal DNA in maternalplasma, increasing the feasibility for clinical application of non-invasive prenatal diagnosis. Methods: 29 maternal blood sampleswere collected from gestation 8-15 weeks with Streck’s Cell-freeDNA BCT tube and cffDNA were extracted by QIAamp CirculatingNucleic Acid Kit. Cell free Fetal DNA content and ratio in plasmawere quantified and estimated by real-time PCR. Results: A totalof 29 plasmas from maternal blood (gestation week ranged from 8to 15 weeks) were analyzed. The total DNA isolated from 5mlplasmas ranged from 337.72 – 1243.88 GE/ml. 16 of 29 maternalplasma samples were Y-positive, from which the Y specificsequences were used to quantify the fetal DNA content. Fetal tototal DNA ratio in plasma ranged from 3.45% to 15.53%, whereasthe mean fetal DNA isolated was 51.80 GE/ml (ranged 23.72 –87.15 GE/ml). The 5ml protocol can recover more than 100 GE offetal DNA (ranged 118.6-435.75 GE) from 10 ml of maternalblood. Conclusions: This workflow enables cffDNA to bepreserved and collected in higher yield than normal collectionmethod. Compare to the literature reported, our fetal to total DNAratio was 2 folds better (i.e. median ranged from 9.21 and 14.06%compared to 4.8% and 4.1% for first and second trimester

respectively). Furthermore the collected Blood sample can bepreserved up to 7 days with no significant change. Therefore, thisworkflow greatly opens up the possibility of performingdownstream application for non-invasive fetal diagnosis.

P050

Quantitative detection of pathogens and ofmRNA/gene mutants: Internal and external referenceRNAs and DNAs for the development and high levelquality & performance controls of real-time RT-PCRand PCR assays

Guido Krupp, Andreas Hanne, Peter Scheinert

AmpTec GmbH, Germany

One important aspect of using synthetic internal control RNAs:false negative results are easily discovered. Availability ofcustomized RNAs allow monitoring of both steps in RT-qPCRtests: the crucial cDNA synthesis and the subsequent PCRamplification. High quality dilution series of positive controls allowroutine implementation of performance control of the tests andguarantee continuous high quality standards. The development ofmutant screening tests is straightforward and any desiredsequence variant is available in homogeneous form, independentfrom the limited availability of (uni-variant) biological samples. Animportant aspect of pathogen detection is the evaluation of testspecificity: only the intended target sequence(s) give positiveresults, closely related species are not detected and give negativeresults. Availability of the corresponding sequence panels can bechallenging for new test developers without resources that havebeen accumulated over many years. The availability of customizedreference sequences offers unlimited possibilities for any facilityand for new fields. Applications of these reference nucleic acidsare demonstrated with instrument platforms from themanufacturers ABI, Qiagen and Roche. Examples are shown forquality & performance control and for the detection of the closelyrelated viral sequences HSV-1 and HSV-2 in one single test.

P051

Validation of appropriate reference genes for thenormalization of real-time quantitative RT-PCR dataobtained from platelets of post-myocardial infarctionpatients

Amir Houshang Shemirani1,2, Katalin Szilvia Zsóri1,2, LászlóMuszbek1

1Debrecen University, Hungary; 2Sátoraljaújhely hospital, Hungary

Human blood platelets contain minute amounts of translationallyactive mRNA. Genetic analysis of platelet mRNA islimited by smallyield of plateletmRNA and the risk of leukocyte contaminationduringplatelet preparation.Previous reports on platelet mRNAexpression have used conventional reference genes, but therehas been no report of studies to check for the validity of thesereference genes. In other words, reference genes had not beenthoroughly investigated in platelet. Method: We isolated leukocyte-depleted platelets from ten mL blood by triple centrifugation. Wetested the reliability, leukocyte contamination, and integration ofplatelet RNA by RT-qPCR to detect specific transcripts.Spectrophotometry (NanoDrop) was used for the qualityassessment of isolated RNA. RT-qPCR was performed onLightCycler® 480. The software NormFinder and geNorm wereused to identify the most suitable reference genes. Expressionprofiles of 9 genes ACTB, EAR, GAPDH, HDGF, GNAS, ANAPC5, OAZ1, EEF2 and CFL1 were established from 20 patients withmyocardial infarction. Results: Nanogram quantities of platelet-specific cDNAs were produced from 10 mL of whole blood. ACTBand HDGF (by geNorm) and ACTB (by NormFinder) were themost stably expressed genes in platelets of patients with thehistory of myocardial infarction. Conclusion: In platelets geneprofiling studies of patients after myocardial infarction the genes ACTB and HDGF in combination are recommended as referencegenes for normalization.


P052

Exogenous RNA control for indication of reactioninhibition in the RT-qPCR workflow

James Covino2, Zaklina Strezoska2, Devin Leake2, AnnaleenVermeulen2, Melissa Kelley2, Jaakko Kurkela1

1Thermo Fisher, Finland; 2Thermo Fisher Scientific, Lafayette,Colorado

Inhibitors of reverse transcriptase quantitative PCR (RT-qPCR)are commonly present in experiments and not easily recognized inexperimental set-ups. The primary challenge is that methods suchas spectrophotometric techniques do not always detect RT-qPCRinhibitors. The Thermo Scientific Solaris RNA Spike Control Kitwas designed as an exogenous control to identify the presence ofreaction inhibitors including those commonly carried throughprocessing steps to isolate RNA such as EDTA, phenol, heparinand EtOH. Here we describe applications for this exogenouscontrol to identify the presence of reaction inhibition and illustratethe impact inhibition can have on results and data interpretation.Experimental results demonstrate RNA Spike Control providesaccurate identification of RT-qPCR inhibition and the ability topredict when inhibition has been removed, for example, by dilutionof the RNA sample into the reverse transcription reaction. TheRNA Spike Control identified RT-qPCR inhibition in a geneexpression assay, explaining the variable results obtained.Furthermore, the use of RNA Spike protocol was compared tocurrent inhibition identification protocols and was found to be aneasy and succinct workflow, offering a clear advantage of thistechnology.

P053

Internal controls for quantification of alternativelyspliced transcripts by real-time PCR

Julia Meiser, Julia Frühwald, Stephan E. Philipp

Institute for experimental and clinical pharmacology andtoxicology, University Saarland, 66421 Homburg, Germany

More than 90% of human genes express mRNAs that undergoalternative splicing resulting in an extensive collection of isoformsthat are required for a huge variability of functions. The ratio of thevariants is tightly controlled and needs to be determined precisely.Real-time reverse transcription polymerase chain reaction (RT-qPCR) is the method of choice to quantify mRNA expressionlevels and has also been adapted to measure the ratio of splicevariants derived from a single gene (1). However, commonly usedassays do not compensate for differences in the reversetranscription efficiencies among the variants and do not includeinternal controls that reveal experimental errors. Here we presentan expanded method to quantify the ratio of alternatively splicedtranscripts by RT-qPCR. In addition to transcript specific primersthe method includes control primers that allow compensation ofvariations during reverse transcription. Furthermore they serve asan independent control of the total number of transcript. Toestimate the PCR efficiencies we used the comparativequantitation tool of the rotorgene software which is based on thedetermination of the second derivative maximum (SDM) of theamplification curve. Comparison of efficiency corrected values tothose obtained by standard curves as well as a new algorithm tocalculate the PCR efficiencies (LinReg, (2)) showed that SDM-data were equally reliable and that standard curves aredispensable. The one-step protocol can be easily adapted to mostsplice events and allows simultaneous identification of twodistinguishable products specific for both transcripts. We showthat the method is applicable to a broad range of templateconcentrations. Furthermore, it is possible to determine minutechanges of the ratio of two variants of ~ 10 %.

1. Vandenbroucke, I. I., Vandesompele, J., Paepe, A. D., and Messiaen, L.(2001) Nucleic Acids Res. 29, E68

2. Ruijter, J. M., Ramakers, C., Hoogaars, W. M., Karlen, Y., Bakker, O.,van den Hoff, M. J., and Moorman, A. F. (2009) Nucleic Acids Res. 37, e45

P054

Contamination control with the heat-labile cod UNGensures accurate and reliable results in your post-PCRanalysis

Elisabeth Lill Andreassen, Olav Lanes, Morten Elde

ArcticZymes AS, Norway

PCR is sufficiently sensitive to detect single copy genes fromsingle cells. Unfortunately, this extreme sensitivity of PCR alsorestricts the method, making it prone to false-positive results dueto contamination. One of the major sources of these false-positiveresults is carry-over contamination of amplification products fromprevious PCRs (1). As the amplicons are both identical to thetarget molecule and produced at a very high copy number duringPCR, they constitute a serious threat, particularly to forensic,diagnostic and palaeogenetic analysis (2). With the advent of theuracil-N-glycosylase (UNG) de-contamination technology, thisproblem became widely solved; however, reactivation of UNGpost-PCR has restricted its use (3,4,5,6). Upon reactivation ofUNG, the newly produced PCR products quickly degrade, thusimpairing downstream analysis such as cloning, sequencing andgenotyping. So the complete and irreversible inactivation of UNGis of pivotal importance for post-PCR applications. In our recentstudies we have demonstrated how a range of commerciallyavailable UNGs fail to meet these criteria. Residual UNG activitywas measured by incubating UNG-treated PCR products at roomtemperature at different time intervals and the extent of DNAdegradation was evaluated on agarose-gels. We furthersequenced the UNG-treated PCR products and evaluated thequality of the sequences. Our study showed that the only UNGthat became completely and irreversibly inactivated by moderateheat-treatment, was cod UNG. The post-PCR sequencing resultsshowed that every UNG tested, except the cod UNG, gave poor orunreadable sequences. Hence, by employing cod UNG as the de-contamination enzyme in PCRs, a range of downstream analysisof PCR products can be performed without the probability of UNG-reactivation impairing the results.

1. Kwok, S., Higuchi, R. (1989); Nature 339:237–238 2. Pruvost, M.,Grange, T., Geigl, E.M. (2005): BioTechniques 38:569–575 3. Longo,M.C., Berninger, M. S., Hartley, J. L. (1990); Gene 83:125–128 4. SobekH., Schmidt M., Frey B., Kaluza K. (1996); FEBS Lett 388:1-4 5. Thornton,C. G., Hartley, J. L., Rashtchian, A. (1992); Biotechniques 13(2):180–1836. Champlot, S. et al (2010) PLoS ONE 5(9):e13042

P055

NOVEL APPROACHES IN MANUFACTURING HIGHQUALITY REAGENTS FOR IN-HOUSE PCRDIAGNOSTICS

Muriel Craynest

EUROGENTEC, Belgium

Background. Emerging infectious diseases like H1N1, haveprompted the need for rapid development of routine PCR testingby service laboratories. The rapid need for such tests requires in-house development and manufacturing. However, likecommercial kits, in-house PCR needs to be built with reagentsofhigh and consistent quality to ensure reliable and accurate assayperformance. Currently, most laboratories will source ‘research-grade’ oligos (produced under ISO 9001 quality system) ratherthan “diagnostic” GMP reagents(produced under ISO 13485) tosave costs. From a quality perspective this is farfromoptimal.Moreover, with more and more medical labs upgrading theirquality system to ISO 15189, control measures over criticalreagents and their suppliers must be implemented. As oligos arethe most critical components of PCR, an innovative manufacturingsolution is proposed in this paper: Clinical Lab Oligos. Methods.FMEA-based risk analysis of the oligo production process wasperformed, followed by appropriate measures to minimize risks fordiagnostic users. Risks were classified as low/medium/high, basedon Severity (impact for customer), Occurrence & Detection.Results. We identified 471 risks in the first round. Using areiterative process, measures were implemented to reduce allrisks to “low” classification. Important measures included, the useof classified clean rooms to eliminate contamination risks, GMPdocumentation (SOP and checklists), and validatedinstrumentation and QC methods. The improved productionprocess is managed according to ISO 13485. Conclusion. Clinicallab oligos will significantly add to further quality improvement of in-house PCR Diagnostics.


qPCR Biostatistics & Bioinformatics

P056

Real-time PCR quantifications in presence of inhibitorsusing SOD and Cy0 methods.

Davide Sisti, Michele Guescini, Renato Panebianco, PasqualeTibollo, Michela Mantuano, Marco Rocchi, Vilberto Stocchi

University of Urbino, Italy

Introduction: Real-time PCR has become the technique of choicefor absolute and relative nucleic acid quantification. The goldstandard quantification method in real-time PCR (Ct) assumes thatthe compared samples have similar PCR efficiency. Recently, thereal-time PCR basic quality control guidelines of quantificationhave been proposed, in order to achieve a reliable quantification.However, in some biological samples, some inhibitors can becopurified with DNA template and often it is hard to remove them.Consequentially, differential inhibition could be observed resultingin real-time PCR quantification bias. In order to avoidquantification inaccuracy, due to differences in amplificationefficiency among run samples to be compared, outlier detectionmethods, based on amplification estimation, have been reported.Recently a new method, namely SOD, showed a high sensitivityand specificity to detect outlier runs. SOD (shape outlier detection)compares shape of amplification curve of experimental runs withstandard curve, without comparing efficiency. Moreover, a newDNA quantification method, called Cy0, show better accuracy inpresence of inhibitors. Hence, we compared DNA quantificationobtained using Cy0 and Ct methods, considering SOD value ofeach run. Material and methods: The DNA consisted of a pGEM-T(Promega) plasmid containing a 104 bp fragment of themitochondrial gene NADH dehydrogenase 1 (MT-ND1) as insert.Real time PCR amplifications were conducted using LightCycler®480 SYBR Green I Master (Roche) according to themanufacturer’s instructions. Ct (fit point method) was determinedby the LightCycler® 480 software version 1.2. The Cy0 value isthe intersection point between the abscissa axis and tangent ofthe inflection point of the Richards curve obtained by the non-linear regression of raw data. Shape based kinetic outlierdetection (SOD) was based on the shapes of the amplificationcurves. SOD value is based on Mahalanobis distance; it is auseful way of determining the similarity of an unknown multivariatesample set to a known one, with multinormal distribution. Results:For each runs, in presence of biological inhibitors such as tannicacid (range: 0.39– 100 mg/mL ), IgG (range: 0.39– 200 mg/mL ) orquercitin (range: 0.31– 40 mg/mL ), Cy0 and Ct values have beencalculated. When estimated DNA concentrations, using Ctmethod, were significantly different from expected values, due toinhibitor presence, SOD efficiently marked these amplificationprofiles as outliers. Instead, DNA quantifications obtained with Cy0method, were not significantly different from the expected values,obtained from residual distribution of curve calibration runs.Conclusion: Our results demonstrated that in presence of threedifferent type of inhibitors at different concentrations, SODefficiently detected outlier runs and Cy0 method achievedquantifications with accuracy significantly higher than Ct method.

Web site: http://www.cy0team.uniurb.it

P057

Kinetic models of qPCR as applied to the problem oflow copy numbers

Alexander M Chagovetz, James P Keener

University of Utah, United States of America

We propose an alternative way of analyzing qPCR data based onkinetic modelling of multiple competing reactions. Three differentmodels are developed for kinetically distinct cases: SYBR green-type detection, hybridization probes, and hydrolysis (TaqMan)-type probes. Instead of analyzing local features of the growthcurve (Ct) we propose a global fit of the growth curve as definedby the model. Our approach demonstrate certain advantages inquantification of low copy number targets, which is espaciallyrelevant for single cell qPCR.

Proposed method allows to:

1. Estimate changes in amplification efficiency during the courseof reaction as a function of initial target concentration.

2. Expand the dynamic range of quantitations and improve itsstability

3. Reliably distinguish positive (non-zero copy number) andnegative (null) samples

P058

Modeling Immuno-qPCR to Estimate Protein Quantity

Harrison Leong, Elana Swartzman, Jennifer Holmdahl


Obtaining relative quantification information from immuno-qPCRassays involves measuring Ct values for serial dilutions ofunknown and reference samples. In general, these curves havelog-linear ranges and, within these ranges, the curves are non-parallel. A theoretical model is developed and used to provide ameans to estimate relative quantity in a way that accounts forthese characteristics. The theory makes it clear that the methodmust be calibrated and suggests several alternatives to achievethat. Experiments have been devised and executed to generatedata with “known” underlying fold change values. A calibrationand fold change estimation approach are cross-validated againstthese data. Results suggest that the method can determine foldchange within a half log.

P059

Development of a GMO screening database withcombinatory algorithms and its application in officialfood and feed control

Lars Gerdes, Sven Pecoraro


The development and cultivation of genetically modified crops isincreasing globally [1]. Food and feed imports from outside the EUwill subsequently require more effort from the responsibleauthorities in monitoring compliance with effective labellingdirectives in the future. Our aim was the development of acombinatory screening system to support an efficient andcomprehensive routine laboratory analysis in Bavaria, Germany.The project consists of two parts: the development of a databasefor collection and interpretation of information related to geneticallymodified organisms (GMO) and the design of ready-to-usemicrotitre plates (modules) for experimental screening of samples.Different genetic elements (e.g. promoters, terminators, structuralgenes) are artificially introduced into plants to establish newgenetic modifications. The introduced elements may vary betweendifferent GMO lines, depending on the intended traits (e.g.resistance to herbicides or insects, changes in metabolitepatterns, etc.). Screening for such inserted elements with (real-time) PCR is a common first step to analyse samples for thepresence of any genetical modification. From the pattern ofdetectable and not detectable elements, valuable conclusionsabout the identity of putative present GMO line(s) can be drawn.Information about selected genetic elements from the literature [forexample 2], applications for authorisation and other (web) sourceswere systematically integrated in a matrix-format. Special carewas taken to additionally record the sources of the information,thus facilitating evaluation of screening results, and tracing ofpossible errors in the matrix. Data from the matrix was accessedwith several implemented algorithms, for example to interprete theoutcome of a screening for genetic elements with the ready-to-usemicrotitre modules, and thus narrow down the candidates forsubsequent identification reactions. Database and screeningmodules are open for implementation of further GMO lines andadditional genetic elements. Together, data processing andcorresponding ready-to-use real-time PCR modules will supportthe efficient analytical food and feed control at the Bavarian Healthand Food Safety Authority (LGL).

1. James, C., Global Status of Commercialized Biotech/GM Crops: 2009.ISAAA Brief, 2010. 41. 2. Waiblinger, H.-U., B. Boernsen, and K. Pietsch,Screening-Tabelle für den Nachweis zugelassener und nicht zugelassenergentechnisch veränderter Pflanzen. Deutsche Lebensmittel-Rundschau,2008. 104(6): 261-4.


P060

Advanced copy number variant analysis withqbasePLUS

Barbara D'haene, Jo Vandesompele, Jan Hellemans

Biogazelle, Zwijnaarde, Belgium

Copy number changes are known to be involved in numerousgenetic disorders. Moreover, copy number polymorphisms ofvarious sizes are thought to contribute to normal phenotypicvariation and susceptibility to multifactorial disease. In this context,qPCR-based copy number screening may serve as the method ofchoice for targeted copy number screening as it has manyadvantages over alternative methods, such as its low consumableand instrumentation costs, fast turnaround and assaydevelopment time, high sensitivity and open format (independentof a single supplier). Here, we present several experiments inwhich we performed targeted deletion screening in patients withhuman genetic disorders. Accurate copy number calling andobjective interpretation was performed with an advanced modulefor copy number analysis integrated within qbasePLUS 2.0. Thesoftware allows the selection of more than one reference samplefor accurate copy number calling. In addition, it provides flexibilitywith regard to the reference samples as these samples may havevarying copy numbers. The identified copy numbers changes arevisualized on a per sample basis and conditional bar colouring isapplied for easy detection of deletions and amplifications. Insummary, we provide guidelines for qPCR-based copy numberscreening and subsequent data-analysis to improve the qualityand reliability of your results.

P061

Full-Process-Kinetics PCR analysis: a holistic modelto PCR data interpretation.

Antoon Lievens1, Stefan Van Aelst2, Marc Van den Bulcke1,Els Goetghebeur2

1Institute of Public Health, Belgium; 2Ghent University, Belgium

Real-time PCR analysis has become a key application inmolecular biology. Yet, despite its general use and markedimprovements in thermocycler hardware over the past decade,little has changed in the way real time PCR data are typicallyanalyzed. Most current approaches assume constant efficiencyover a single reaction. Such methodology performs well as long aslong as PCR efficiency does not significantly vary betweenreactions. Yet, small inaccuracies in reaction efficiency can lead tolarge quantification errors. Especially when working with biologicalsamples, the possible presence of coextracted inhibitors poses achallenge to data analysis. A new approach to modeling real-timePCR data is presented, called Full Process Kinetics PCR (FPK-PCR). It combines a kinetically more realistic model with flexibleadaptation to real time PCR data. The approach aims atreconstructing the entire chain of cycle efficiencies, rather thanjust the data in a limited 'window of application'. As a result, oneextracts additional information and loses a level of arbitrariness.The method reveals the amplification steps underlyingfluorescence increases. This opens access to detailed informationabout the internal efficiency behavior and derived parameters ofinterest. The novel approach was tested on real time SYBRgreenPCR data originating generated with genetically modified roundupready soybean (event GTS 40-3-2) genomic DNA. Addition of aknown PCR inhibitor, isopropanol, allowed to evaluate themethods ability to distinguish inhibited from non inhibitedreactions. Results: Efficiency estimates returned by the model arecomparable in both accuracy and precision to the golden standardof serial dilution on the one hand, and to other single reactionefficiency calculation methods on the other hand. The changes inefficiency throughout the reaction as described by the FPK-PCRprocedure are however considerably closer to the actual data thanother models available through the literature. The FPK-PCRapproach further allows to identify inhibited reactions by correctlyestimating maximal reaction efficiency.The approach thusdemonstrates that by implementing a global efficiency model onecan avoid the selection of a window of application. Wheninvestigating gene expression or quantifying sample content,differences in efficiency between samples before and aftertreatment, or between a sample and a standard, introduce biasthat may skew the conclusions of an assay. FPK-PCR now allowsto avoid such bias by yielding efficiencies that are

comparablebetween reactions. Knowledge of the individualreaction efficiencies may be used to compensate for efficiencydifferences between reactions or to simply remove reactions withaberrant efficiency from analysis. Finally, the individual cycleefficiencies enable reconstruction of the fluorescence data,providing a quality control over the efficiency estimate.

High Throughput qPCR & digital PCRNext Genartion Sequencing

P062

Identification of Tumor Cell Lines by MolecularSignature using Digital PCR and TaqMan® CopyNumber Assays

Julie Kuykendall


Digital PCR (Polymerase Chain Reaction) is an advancedutilization of PCR technology where the number of nucleic acidtemplate is accurately counted. The digital PCR technology relieson segregation of individual target template into differentreactions. This can be achieved by diluting the nucleic acidtemplate to the Poisson limit where in each PCR reaction containseither a single copy of template or none. Then PCR amplificationis performed where the reaction wells contains template will showamplification signal while the wells without template shows nosignal. The 1 and 0 scoring of the wells gives the digital nature ofthis PCR practice. The positive wells are counted and the numbercan be extrapolated to exact starting number of template. Toachieve statistically accurate counting by digital PCR the numberof replicates needs to be quite large. Thousands of replicates arenecessary to achieve greater than 95% confidence in the countednumber. Chromosomal rearrangement is a common feature intumor derived cell lines. Here, we describe an approach usingdigital PCR to detect chromosomal aberration in tumor cell lines.We use 48 TaqMan® Copy Number Assays to count the copynumber of chromosomes. Separately, we use the TaqMan® assaytargeting the RNaseP gene on chromosome 14 as a copy control.We counted genomic DNA extracted from 6 different humancancer cell lines (A431- epidermoid carcinoma, HeLa- cervicaladenocarcinoma, Raj- B cell lymphoma, K562- chronicmyelogenous leukemia, Jurkat- acute T cell leukemia, MCF7-breast adenocarcinoma). We also used genomic DNA normalhuman cell lines (one each female and male) as controls. Wefound chromosome numerical aberration throughout the entiregenome for all the human cancer cell lines. Some chromosomesare duplicated entirely where all 4 copy number assays show thesame count. While some chromosome are missing entirely wherethere were no signal from all 4 assays targeting the samechromosome. The results are consistent with the aneuploidynature of these tumor derived cell lines. There are also sectionalduplication and deletions within chromosome where the 4 assaystargeting the same chromosome show different counts. Thecombination of chromosome numerical aberration and intrachromosome rearrangement pattern is unique to each cell line.This allows the identification of cell line by digital PCR signature.The utilization of digital PCR for nucleic acid template countingenables accurate determination of starting template. Furthermorethe absolute numerical result of digital PCR counting methodallows direct comparison between different results obtained fromdifferent run or instruments. This is an advantage to thresholdcalculated result from traditional qPCR where within-runnormalizers are required to compare between runs andinstruments. The use of digital PCR is a significant evolutionarystep in nucleic acid quantification method.


P063

High throughput microfluidic dynamic array profilingdemonstrates differential gene expression of immunerelated genes during early pregnancy in cattle

Anna E Groebner1, Katy Schulke1, Horst D Reichenbach2,Heinrich HD Meyer1, Susanne E Ulbrich1

1Physiology Weihenstephan, Technische Universitaet Muenchen,Germany; 2Bavarian State Research Center for Agriculture,Institute of Animal Breeding, Grub, Germany

Due to the genetic disparity of the semi-allogenic embryo and themother, potential tolerance inducing genes are required for theestablishment of pregnancy. To determine the transcriptabundance of immune relevant genes in estrus synchronizedpregnant and non-pregnant Simmental heifers, total RNA wasextracted and reverse transcribed from ipsilateral endometrialtissue (days 12, 15 and 18 after onset of estrus) and pre-implantation conceptuses (days 15 and 18). The high throughputgene expression platform (Fluidigm® 96.96 Dynamic Array) wasused based on microfluidic dynamic arrays allowing 9´216 realtime PCR gene expression measurements per chip. The mRNAexpression of 32 genes in 94 preamplified endometrial andembryonic samples each was measured with three replicates on asingle chip. We obtained an average standard deviation of 0.16and a chip to chip correlation of >0.99 for all three replicates. Theplatform thus allows a rapid large scale analysis of real time PCRassays with a high dynamic range and sensitivity. Proinflammatorycytokines analyzed were not differentially expressed. However, asignificant 18-fold (p<0.001) higher transcript abundances forindoleamine 2,3-dioxygenase 1 (IDO1) in the endometrium ofpregnant vs. non-pregnant animals was detected. IDO1 encodesfor the initial L-tryptophan degrading enzyme of the kynureninepathway and is known to inhibit activity of lymphocytes.Furthermore, β2-microglobulin (B2M), the small subunit of MHCclass I molecules was only marginally expressed on conceptustissue when compared to the endometrium, thus the conceptusmight avoid immunological detection by the maternal immunesystem. In conclusion, our data point towards an interaction ofvarious tolerance inducing strategies at the embryo-maternalinterface required for the establishment of pregnancy.

Supported by DFG (Ul 350/1-2, FOR 478)

P064

RealTime ready qPCR Assays - Workflow and aninterim census of 10k+ functionally tested qPCRassays

Christine Bruns, Manuel Dietrich, Ursula Grepl, Rita Haerteis,Ingrid Hoffmann, Regina Huber, Bastian Kramer, IreneLabaere, Ralf Mauritz, Elke Stadlbauer, Heiko Walch

Roche Applied Science, Germany

On the RealTime ready (RTr) configuration portal we offer pre-tested human, mouse and rat qPCR assays based on the uniqueUniversal ProbeLibrary technology. The main advantage of RTrqPCR assays is that each individual assay is functional tested forspecificity, PCR efficiency, signal intensity etc. according tostringent criteria and in general consistency with the recentlypublished MIQE guidelines. Here we provide a detailed overviewof the involved processes for the RTr on-demand qPCR assaydevelopment. The first step involves using the comprehensivesearch functionalities provided within the RTr Configurator toidentify the target gene of choice from one of the three organisms.The in-house development workflow starts by defining appropriatetranscripts for individual targets, continues with design, testing andassay selection procedures and concludes with the final assayproduction and shipment either as single liquid assays or asmultiple dried-down assays in 96/384 well plates. All individualprocess steps are described, summarizing the generalspecifications as well as the experience gathered from 10k+successfully developed qPCR assays.

P065

Parallel Preparation of Targeted ResequencingLibraries from 480 Genomic Regions Using MultiplexPCR on the Access Array™ System

Fiona Kaper1, Jun Wang, Megan J. Anderson, Peilin Chen,Min Min Lin, Martin Pieprzyk, Robert C. Jones, Andrew P. May

Fluidigm Corporation, 7000 Shoreline Court, Suite 100, South SanFrancisco, CA 94080

Next generation sequencing platforms have dramatically reducedsequencing costs. However, it currently remains too expensive toroutinely resequence entire human genomes in order to discovergenetic variants or somatic mutations underlying tumorigenesis.Therefore, a need exists for multiplexed, targeted amplificationmethods that allow for the analysis of multiple genomic regions inlarge cohorts. Available targeted enrichment technologies areeither aimed at the capture of regions of interest from a singlesample, exhibit uneven representation or require significantamounts of input material. The novel microfluidic platform, theAccess Array™ system, combines 48 samples with 48 primer setsresulting in 2,304 simultaneously occurring PCR amplificationsrequiring as little as 50ng DNA per sample. PCR productsgenerated on the Access Array™ system can be used forsequencing on all next-generation sequencing platforms, including454 GSFLX and Illumina GAII. To increase coverage andthroughput, PCR reactions can be multiplexed within Access ArrayIFCs generating up to 480 amplicons per sample.

P066

Integration of a novel real-time PCR detection systemonto Luminex beads in a PCR reaction.

Ross Thomas Barnard1, Richard Lai1, Simon Corrie2, Che-Cheng Yeh1, Graeme Barnett3, John Feros3

1School of Chemistry and Molecular Biosciences,The University ofQueensland, Australia; 2Australian Institute for Bioengineering andNanotechnology, The University of Queensland; 3BioChipInnovations Pty. Ltd., Suite 4, 1407 Logan Rd, Mt Gravatt, Qld4122, Australia

We previously reported the development of a new real-time PCRdetection system, (PrimRgloTM), that utilises “tagged” PCRprimers, a fluorophore labelled ‘universal’ detectionoligonucleotide and a complementary quenching oligonucleotide(1,2). In that solution phase embodiment, the fluorescence signaldecreased as PCR product accumulated due to the increase indetection/quencher hybrid formed as the tagged primer isconsumed. We used influenza A matrix gene and porA and ctrAgenes of Neisseria meningitidis as targets for developing thatsystem. Ct values were generated and the sensitivity of thePrimRglo system compared very favorably with the SYBR greenand TaqmanTM detection systems (1). In a new development, thePrimRgloTM system has now been integrated with LuminexTM

microbeads, utilizing a universal capture oligonucleotide coupledto the microbead, and a Cy3 label on the universal detectionoligonucleotide. PCR (target: influenza A matrix gene plasmid)was carried out, with the oligonucleotide-conjugated Luminexbeads in the PCR tube throughout the PCR reaction. The PCRreaction mix was injected into the Luminex machine at the end ofthe reaction. Reactions were sampled at different cycle numbersand the Cy3 fluorescence signal associated with the microbeadsincreased in proportion to the amount of PCR reaction product.The Luminex machine currently has the capacity to measure 96different reaction products attached to optically distinguishable

bead sets, so the integration of the PrimRglo real-time detectionsystem onto Luminex beads has the potential to enable highdegree multiplexing of real-time PCR reactions, beyond that whichis currently possible with standard methods.

1 . R. Barnard, R. Lai, D. Pearson, Z.Y. Phua, D. Whiley, T. Sloots and G.Barnett. A new multiplexable, quantitative, real-time system for detection ofnucleic acids. New Biotechnology, Volume 25, Supplement 1, September2009, Pages S17-S18, abstracts of the 14th European Congress onBiotechnology, Barcelona, Spain 13–16 September, 2009.

2. Barnard, R.T. & Barnett, G.R. (2007) A method and kit for analyzing atarget nucleic acid sequence. WO 2007/003017.


P067

Performance and Accuracy Breakthroughs with the5500 Series SOLiD™ Sequencers

Oliver Stephan1, Timothy Burcham1, Patrick Kinney1, SteveBoege1, Jon Hoshizaki1, Matt Chan1, David Cox1, JasonBriggs1, Lee Jones1, Janet Ziegle1, Larry Joe1, Sylvia Chang1,Kathleen Perry1, Takuya Matsui2, Shuhei Yamamoto2, RyojiInaba2

1Lifetechnologies, Inc; 2Hitachi High Technologies, Hitachinaka-shi, Ibaraki-ken, Japan

The 5500 Series SOLiD System incorporates a number ofimprovements in system design and chemistry to give the highestand most accurate throughput of any next generation sequencer.The 5500 series uses a 6 lane FlowChip™ where clonally-amplified beads are deposited. The 5500xl supports twoFlowChips while the 5500 supports one FlowChip. Each FlowChiplane is independently addressed with a proprietary, direct-injectionfluidics system which decreases run time, minimizes dead-volumeand greatly simplifies the design. Each lane can contain a distinctsample: The system will deliver the required reagents to eachlane, independent of the other lanes, using only the reagentsrequired. Imaging time on the 5500 is made faster byincorporating an innovative fast filter wheel, hardware autofocus,and fast camera. Images are processed in real time using aworkstation-class computer utilizing state-of-the-art algorithms andGPGPU. The amount of data generated and saved is reduced byan order of magnitude. Chemistry improvements include longer 75bp reads (forward), 35 bp (reverse paired end), and 2x60 longmate pair. Accuracy improvements using the Exact CallChemistry™ system increase accuracy to 99.99% allowingheterogeneous SNP detection at levels requiring much lessredundancy and improved confidence levels. Users can expectdata rates of 20-30 Gb/day with the 5500xl. Results from severallibrary types and applications will be shown, including run times.Pre-release results using Nanobeads will also be shown with datarates >40 Gb/day and typical throughput of greater than 350Gb/run.

P068

qPCR in immune system monitoring : a highthroughput method

Maxime Lilian Dooms, Flore Depeint, Afif M Abdel Nour


The impact of food consumption on the immune system is fastbecoming a major target for human nutrition, especially in the areaof health claims. When developing methods for application inclinical trials, major concerns are cost effectiveness and subjectwell being. Based on these criteria, we developed a method usingRT-qPCR leading to repeatable data in less than 48 hours. Humanwhole blood is collected during the dietary intervention trial andchallenged in vitro with lipopolysaccharide (LPS), resulting in aninflammatory reaction and a shift in expression of immunemarkers. This shift can be measured using RT-qPCR performedon RNA extracted after incubation. Relative expression of thetarget gene for each subject is normalized against a set ofreference genes. Solaris (Thermo Scientific) specific primer andprobes targeted to genes of interest, such as cytokines TNF alphaor IL6 are used to quantify genes expression. Those primers andprobes combinations target shorter areas than usual, allowing awider choice in amplicon location, while keeping meltingtemperature constant. The use of one-step protocols, ready to useoptimised mixes including primers and probes, reduces the timerequired for setting up a qPCR as well as risk for handling failures.

P069

Transcriptome analyses in rice: validation ofmRNAseq using qRT-PCR

Tina Kyndt1, Geert Trooskens1, Simon Denil1, Tim Demeyer1,Annelies Haegeman1, Wim Van Criekinge1,2, GodelieveGheysen1

1Department of Molecular Biotechnology, UGent, Belgium;2NXTGNT, UGent, Belgium

The transcriptome represents a comprehensive set of transcribedregions throughout the genome. Studying transcriptome dynamicsprovides important insights into the functional elements of thegenome, their expression patterns and the regulation oftranscribed regions. Recently, Next Generation Sequencingmethodologies have been introduced into transcriptome research,with mRNAseq currently being the most innovative and high-throughput method for expression profiling. Next to comparativegene expression analyses, mRNAseq can be used for discoveringthe full extent of 5' and 3' untranslated regions, novel splicejunctions, novel transcripts, alternate transcription start sites, andrare transcripts. The goal of our research project is to applymRNAseq to study the transcriptome of rice roots whenchallenged by nematode infection. Rice has been intensivelystudied in the past decades, since it is one of the most importantcrop plants worldwide and an excellent model system formonocotyledonous plants. The rice root knot nematodeMeloidogyne graminicola and the migratory root rot nematodeHirschmanniella oryzae are two agronomically important ricepathogens with very different lifecycles. Even though they causesubstantial harvest losses in rice growing areas throughout theworld, almost no fundamental research has been performed onthese nematodes and their interaction with the rice plant.Comparing the transcriptome in rice upon infection with thesepathogens will provide important insights into general and specificdefence strategies of the rice plant towards nematodes. To testthe robustness of mRNAseq for identifying differential expressionin plant tissue, the transcriptome of uninfected rice roots and roottips was first evaluated. On average 20 million reads per samplewere generated (76 nt reads, single end), they were mapped ontothe rice genome and differential expression was evaluated usingBayseq. Our analysis uncovered over 2000 DEGs (differentiallyexpressed genes), of which 1800 showed higher expression in theroots, whereas 390 are overexpressed in the root tips (at an FDRcut-off of 0.05). A subset of these DEGs were independentlyvalidated using qRT-PCR analysis, all of which confirmed thedifferential expression pattern in rice roots and tips obtained bysequencing.

Single-cell qPCRqPCR from limited material

P070

At the single cell level cellular noise challengesnormalization

Richard Fekete, Ron Abruzesse, Elena Grigorenko


As expected, biological systems such as tissues exhibitconsiderable phenotypic variation. Surprisingly, variation evenoccurs in “clonal” populations. Until now this heterogeneity hasbeen attributed to differences in cell cycle, microenvironments, orcell type. These differences would be expected to affect geneswithin a cell in a similar manner, i.e. cells at the same point in thecell cycle would have the same gene profile. However, it has nowbeen shown that there are also differences within identicalbiological processes and this has been referred to as “noise”.These differences likely result from stochastic events within a celldue to the limited numbers of components such as RNApolymerases. To look at the effect of noise on gene expression ina large number of genes we used the Cell Sensor AP-1-bla ME-180 cell line as a model system. We treated cells with epidermalgrowth factor to stimulate the RAS and MAPK pathways andchose target genes predicted to be up- and down-regulated. Inorder to analyze gene expression profiles from a large number ofindividual cells we used the Single Cell-to-Ct kit. This kit includesoptimized reagents for sample preparation, reverse transcription,preamplification and qPCR. We compared normalized expressionlevels of a number of target genes (normalized to a variety ofcontrol genes), with non-normalized expression levels. We thencompared this data to samples of cells prepared en masse. Wefound that for samples prepared en masse, gene expressionlevels varied from sample to sample. However, expression levelsof all genes changed equally between samples. This allowedsample to sample differences to be eliminated when target geneswere normalized to control genes. When analyzing single cells we


also found that expression levels of genes varied from cell to cell,however, in contrast to cells analyzed en masse, expression of allgenes did not change equally from cell to cell. This caused thenormalized expression profiles to vary widely from cell to cell, andchange depending on which genes were used to normalize. Byusing a digital approach to analyze RNA expression profiles wedemonstrate that gene expression from single cells is affected bymany factors that are not relevant to the analogue way ofanalyzing cells en masse. This study challenges the wellunderstood field of gene expression, which routinely uses controlgenes to normalize expression profiles and demonstrates thatunderstanding expression profiles from single cells will require adifferent method of analysis and presentation.

P071

Two different astrocytes subpopulation in cortex ofmice

Vendula Rusnakova1, Pavel Honsa2, Miroslava Anděrová2,Mikael Kubista1

1Institure of Biotechnology, Czech Republic; 2Isntitute ofExperimental Medicine

We classified two astrocytic populations in the cortex ofGFAP/EGFP mice, high response and low response astrocytes,which differ in the extent of cell swelling during the 20 minuteoxygen-glucose deprivation. Based on the pharmacological studywe hypothesize that the existence of two astrocytic population inthe cortex of GFAP/EGFP mice might result from a distinct activityor expression pattern of excitatory amino acid transporters(EAATs), Na+, K+, Cl- cotransporters, volume regulated anionchannels or two pore potassium channels. We performed geneexpression profiling of 180 single cell astrocytes to see differentgroups, 8 different genes were analysed. We found two differentgroups of cells by using three independent different statisticalapproaches (Principal component analysis, hierarchical clustering,Kohonen self organizing map). We identified two subpopulationsof astrocytes with distinct gene expression profiles.

P072

Gene expression analysis of formalin-fixed paraffinembedded laser microdissected endometrial tissuesections

Anna E Groebner1, Nevena Kirova1, Harald Welter2, ArturMayerhofer2, Heinrich HD Meyer1, Susanne Ulbrich1

1Physiologie Weihenstephan, Technische Universitaet Muenchen,Germany; 2Institute for cell biology, Ludwig Maximilian UniversitaetMuenchen, Germany

Establishment of pregnancy is dependent on an accurate maternalreaction towards the ruminant pregnancy recognition signalinterferon-tau (IFNT). To determine cell type specific responses ofinterferon stimulated genes, mRNA expression ofmyxovirusresistance 2 (Mx2) and indoleamine 2,3-dioxygenase(IDO) was analyzed in microdissected formalin-fixed, paraffin-embedded (FFPE) endometrial samples from pregnant and non-pregnant heifers. One mm2 of luminal (LE) and glandularepithelium (GE) of the Zona functionalis (ZF) and -basalis (ZB), aswell as stroma of ZF and ZB from bovine endometrium (n=3,respectively) were collected via laser microdissection (LMD, LaserMicrobeam system, P.A.L.M. Microlaser Technologies). Total RNAwas extracted (RNeasy FFPE, Qiagen), reverse transcribed(MMLV reverse transcriptase, Promega) and preamplified(TaqMan® PreAmp Kit, AppliedBiosystems). Appropriatereference genes for normalization were chosen using GenEx ProVer 4.3.4 (multiD Analyses AB). In pregnant animals, hightranscript abundance was detected for the interferon stimulatedgene Mx2 in every cell type analyzed, showing a mean of5.3 ± 1.4 ΔCq ± SEM in pregnant vs. 1.9 ± 0.6 ΔCq in non-pregnant endometrium. Gene expression for IDO increasedlikewise 11-fold (-0.5 ± 1.4 vs. 2.99±1.4 ΔCq). Interferon regulatory factor 2 (IRF2) expression differed neither in LE (mean-0.85 ± 0.34 ΔCq) nor GE (ZF) (mean 0.001 ± 0.19 ΔCq) with respect to the status. On the contrary, a differential spatialexpression pattern with higher abundance in stroma cells (ZF) ofpregnant animals (-1.4 ± 0.8 ΔCq) than in non-pregnant animals(0.8 ± 0.13 ΔCq) was observed for IRF2. This is in contrast to thepregnant ewe, were expression of Mx2 increases primarily in the

LE and superficial GE (ZF), while the IRF2 is not expressed instroma cells. Thus, gene expression analysis on FFPE LMD tissuesections points to species specific spatial differences in geneexpression during preimplantation.

Supported by DFG (Ul 350/1-2, FOR 478)

P073

Assessing heterogeneity at a single cell level in aclonal population of transformed epidermalkeratinocytes

RM Teisanu1, H Richter2, J Moore3, A Paillusson2, OHaglebüchle2, J Weber2, K Harshman2, Y Barrandon1

1Laboratoire de Dynamique des Cellule Souches, Institute ofBioengeneering, Ecole Polytechnique Fédérale de Lausanne;2Lausanne Genomics Technologies Facility (GTF), Center forIntegrative Genomics, Université de Lausanne; 3FluidigmCorporation, South San Francisco, CA USA

Human epidermal keratinocytes can be extensively cultured invitro, resulting in the formation of epithelial colonies maintained bya stem cell hierarchy similar to that of normal epidermis. Due totechnical limitations, efforts to determine the molecular identity ofepidermal stem cells and their progeny generally rely onassessing gene expression levels in large populations of cells.The major caveat of this approach is the averaging of geneexpression in a functionally non-homogenous population of cells,thus preventing the identification of individual signatures. Thepurpose of this study was to optimize methods that will be furtheremployed in determining gene expression signatures of epidermalstem cells and to determine the level of heterogeneity in a clonalpopulation of immortalized human epidermal keratinocytes (GMA24). The expression level of 32 genes involved in proliferation,differentiation and stemness maintenance was determined inindividual GMA24 cells bearing a fluorescent reporter of the S, G2and M phases of the cell cycle (the fluorescence ubiquitination cellcycle indicator (FUCCI)). To this end we employed a noveltechnology based on microfluidics chips (Fluidigm’s BioMarksystem) that allows the assessment of 96 genes in 96 individualsamples. Data analysis allowed the classification of genesaccording to their variability from cell to cell in low, intermediateand high variability genes. Non-expressed genes were notincluded in the analysis. According to this classification, stablegenes represented 30.4% of the expressed genes. Importantly,56.5% belonged to the highly variable genes, demonstrating thatdespite the clonal origin of these cells, considerable geneexpression variability exists at single cell level. Further studies arerequired to assess whether this heterogeneity results in functionaldifferences between cells. In conclusion, novel technologyenabling the simultaneous analysis and comparison of geneexpression in single cells reveals a previously unrecognizedheterogeneity in a clonally derived population of cells.

P074

Gene Expression Profiling of Duodenal BiopsiesDiscriminates Celiac Disease Mucosa from NormalMucosa

Hanna Bragde1, Ulf Jansson2, Ingvar Jarlsfelt1, JanSöderman1

1Division of Medical Diagnostics, Ryhov County Hospital, Sweden;2Department of Pediatrics, Ryhov County Hospital, Sweden

Objectives: Specific histopathologic changes in the small intestine,which normalize during a gluten-free diet, are characteristic forceliac disease. The histopathologic assessment of these changesin duodenal biopsies is usually routine, but can be problematic. Inthis study we investigated gene expression profiling as adiagnostic tool for celiac disease as well as for monitoringremission upon gluten free diet. Methods: 109 genes wereselected to reflect features that are characteristic of celiacdisease, i.e. alterations in crypt-villi architecture, inflammatoryresponse, and intestinal permeability. Selected genes wereexamined for differential expression in normal mucosa comparedto celiac disease mucosa (histologically graded according to themodified Marsh scale) in pediatric patients investigated for celiacdisease. The ability to classify biopsies using gene expressionwas addressed by means of discriminant analysis. Results: Fiftygenes were differentially expressed, of which eight genes


(APOC3, CYP3A4, OCLN, MAD2L1, MKI67, CXCL11, IL17A, andCTLA4) discriminated normal mucosa from celiac disease mucosawithout classification errors using leave-one-out cross-validation(n = 39), and identified the degree of mucosal damage, dividedinto groups Marsh 0, Marsh 2 - 3B and Marsh 3C. Validation usingan independent set of biopsies (n = 27) resulted in four discrepantcases. Biopsies from two of these cases showed a patchydistribution of lesions, indicating that discriminant analysis basedon single biopsies failed to identify celiac disease mucosa. In theother two cases, serology (levels of anti-gliadin and anti-tissuetransglutaminase antibodies) supports class according todiscriminant analysis and histologic specimens were judgedsuboptimal but assessable. The posterior probabilities of a singleMarsh 1 specimen were strikingly distributed between the Marsh 0class and the Marsh 2 - 3B class, thus suggesting the possibility ofdistinct classification. Conclusion: Gene expression profilingshows promise as a diagnostic tool and for follow-up of celiacdisease. A classification based on gene expression analysis ofthree biopsies per patient would entail a laboratory service costcomparable to that of a histopathologic assessment. Theclassification functions, however, need to be established using alarger cohort of patients, and further validated.

P075

Allele specific gene expression from single cell RNA-seq

Thomas Rygus1, Catalin Barbacioru4, Fuchou Tang2,3, EllenNordman4, Kaiqin Lao4, M. Azim Surani2

1Life Technologies, Germany; 2Wellcome Trust/Cancer ResearchUK Gurdon Institute of Cancer and Developmental Biology,University of Cambridge, Tennis Court Road, Cambridge, CB21QN, UK; 3BIOPIC, School of Life Sciences, Peking University,Beijing, 100871, China; 42Genetic Systems, Applied Biosystems,part of Life Technologies, 850 Lincoln Centre Drive, Foster City,CA 94404, USA

The stochastic gene expression has the potential to determine thephenotype of a cell. However it has never been globally shown inmammalian cells in vivo under physiological conditions. Usingsingle cell RNA-Seq analysis, here we proved that up to 2,000genes in individual cells of the mouse early embryos show allelicdifferential expression. This is compatible with the fact thatregulation feedback by the protein product on its own transcriptionusually can not discriminate between the two alleles of a samegene, which will not damper the differential expression of the twoalleles. For all of these genes, their allelic differential expression isat least partially determined by genetic polymorphisms since oneof the two alleles of the same gene always express lower than theother one in both blastomeres from the same two-cell embryo.This indicates that the subtle genetic polymorphisms between thetwo alleles of a same gene can definitely determine the functionalreadout of the gene in cis through either directly regulate thetranscription machinery or indirectly regulate the epigenetic statusof them in a same cell. At the same time, 1.6% of genes (or up to200 genes) show allelic specific stochastic expression, that is, theexpression ratio between the two alleles of the same gene areclearly different in the two essentially identical blastomeres of thesame two-cell embryo. This proves that stochastic geneexpression exists in mammalian early embryos, which can affectexpression of up to hundreds of genes significantly, and thecombination of deterministic and stochastic expression of thegenes will potentially regulate the precise fate, developmentalpotential, and phenotype of a cell.

HRM & Methylation Studies

P076

The EpiQ Chromatin Analysis Kit: A New Tool forEpigenetic Research

Viresh Patel

Bio-Rad Laboratories, United States of America

Eukaryotic DNA can exist in two general states, euchromatinwhere the DNA is loosely packaged, accessible andtranscriptionally competent and heterochromatin where the DNA istightly packaged, inaccessible and transcriptionally silent.Epigenetics controls the transition between these two states.There are two main epigenetic events, DNA methylation andhistone modification which help regulate the chromatin state andmost current epigenetic analyses study either DNA methylation orhistone modification events. We have developed the EpiQ assay,a new and fundamentally different product that assesseschromatin structure in a quantitative manner and interrogates thefunctional consequence of epigenetic events.

P077

Chloroplast DNA heterogeneity detection with HRManalysis

Irem Uzonur, Esma Ozsoy, Zeynep Katmer, Fatih Koyuncu,Gamze Akdeniz, Ali Rıza Atasoy

Fatih University, Turkey

Chloroplast DNA (cpDNA) has highly conserved regions and alsointergenic spacers with relatively high levels of polymorphisms forthe analysis of intraspecific to intergeneric levels of variation.Chloroplast DNA (cpDNA) is a source of original markers veryuseful in phylogeny and in population genetics. The degree ofuniversality of cpDNA primers within the plant kingdom varies. Inour case primers (KLR-F: 5’-AGTTCGAGCCTGATTATCCC-3’andKLR-R: 5’-GCATGCCGCCAGCGTTCATC-3’) amplify a 298 bphighly conserved region that is virtually a conserved region fromany land plant and many alga with end-point detection. We furtherour end-point detection using qPCR assisted with HRM analysisand sequencing to detect the genetic heterogeneity of cpDNA thatis undetectable with end-point PCR and agarose gelelectrophoresis from terrestrial plants: Urtica dioica, Zea mays,Phaselous vulgaris and bloom forming micro-algae speciesProrocentrum minimum and Skeletonema costatum. In thiscontext as approved by sequencing, HRM assisted qPCR is a verypractical and sensitive tool that can be adapted to the differentlevels of genetic heterogeneity determinations.

P078

Semi-quantitative analysis of O6-methylguanine-DNAmethyltransferase (MGMT) promoter hypermethylationby high resolution melting (HRM) real-time PCR

Kok-Siong Poon1, Lily Chiu1, Evelyn Siew-Chuan Koay1,2

1Molecular Diagnosis Centre, National University Hospital,Singapore; 2Department of Pathology, Yong Loo Lin School ofMedicine, National University of Singapore

Objectives: O6-methylguanine-DNA methyltransferase (MGMT)promoter hypermethylation has been reported as a goodprognostic biomarker for predicting favorable outcome in patientswith glioblastomas on alkylating agent chemotherapy. A reliableprognostic test is able to accurately determine the extent ofpromoter site methylation which is associated with epigeneticsilencing of the gene. Most commonly used methylation-specificPCR assays are based on methylation-specific primers binding toand interrogating the methylation status of a limited number ofCpG sites within the promoter sites of the genes. A high resolutionmelting (HRM) real-time PCR has been developed on theLightScanner 32 system, to serve as a rapid and inexpensivemethod to detect and semi-quantify the full methylation of targetedCpG sites in the MGMT promoter region. Methodology: To createmethylated and unmethylated controls, Cp Genome UniversalMethylated DNA (CHEMICONTM, Millipore Corporation, USA) andgenomic DNA prepared from lymphocytes were bisulfite-convertedwith EZ DNA Methylation-Gold KitTM (Zymo Research, CA, USA),


respectively. The methylated DNA (metDNA) was mixed withunmethylated DNA (unmetDNA) in metDNA:unmetDNA ratios of80%, 60%, 40%, 20% and 10%. Amplification was performed witha set of methylation-independent primers in the LS Mastermix withincorporated LC Green dye (Idaho Technology Inc., UT, USA),and the PCR assay conditions were optimized. The extent ofmethylation of the 108bp amplicon, which encompasses 12 CpGsites in the MGMT promoter region, was determined using theproprietary HRM LS32 Amplicon Genotyping software. Sevenhuman cancer cell lines, namely MCF10A, BT549, SKBR3,SW480, BT474, WM3899 and MCF12A were evaluated for theirMGMT methylation status using this assay. By using the meltprofiles of the above-mentioned methylation standards asreference, the ratios of metDNA to unmetDNA were semi-quantifiable within the intervals of <20%, 20%-40%, 40%-60%,60%-80% and >80%. Results: The 100% fully methylated and100% unmethylated DNAs controls exhibited visuallydistinguishable melt peaks at 80°C and 85°C, respectively. Thenormalized melting curves showed a reproducible dose-responserelationship for different percentages of fully methylated DNA. Fiveof the evaluated cell lines (MCF10A, BT549, SKBR3, BT474 andMCF12A) did not show any methylation; the remaining two(SW480& WM3899) showed positive results with 40-60% metDNAand 100% metDNA, respectively. The assay can consistentlydetect as low as 10% of metDNA in a mixture with 90%unmetDNA. Conclusions: The HRM assay allows the rapidscreening and semi-quantification of full methylation of 12 targetedCpG sites in the MGMT promoter region.

P079

Application of High Resolution Melting to SNPgenotyping of Porcine Ryanodine Receptor Gene

ANNA CASTELLO1, ANNA MERCADE2, ARMAND SANCHEZ1,2

1Departament de Ciencia Animal i dels Aliments, UniversitatAutonoma de Barcelona, 08193 Bellaterra, Spain; 2ServeiVeterinari de Genetica Molecular, Facultat Veterinaria, UniversitatAutonoma de Barcelona, 08193 Bellaterra, Spain

High Resolution Melting Analysis (HRM) is a recently developedtechnique for fast and cost-effective SNP genotyping based on theanalysis of the melting profile of PCR products, using dsDNAintercalating dyes to monitor the reduction of fluorescence thatoccurs from unmelted to melted DNA. A single nucleotidepolymorphism (SNP) in the porcine ryanodine receptor (ryr1) genewas associated with malignant hyperthermia (MH) disease. Thisdisease causes important losses in swine industry due tomanifestation of MH post-mortem signals in susceptible animalsthat affect detrimentally meat quality. As ryr1 mutation plays a keyrole in animal production, it is of main importance to implement afast and economical method of diagnosis. An HRM protocol hasbeen developed to genotype the C/T substitution at nucleotide1843 of ryr1 gene. With the EcoTM Real-Time PCR System andusing the high sensitive “release-on-demand” dye EvaGreen® wehave analysed DNA samples extracted from swine blood withdifferent methods in order to test the effects of genomic DNAquality on melting behaviour. As it is well-known, inhibitorscarryover from the template could result in low sensitivity, poorreproducibility and incorrect genotype. To validate resultsobtained, all tested samples were previously genotyped bypyrosequencing. The HRM technique presented in this study toryr1 genotyping is sensitive enough to allow the detection of theC/T transition. Furthermore, DNA quality obtained with thedifferent extraction methods tested has not been a limiting fact.Obtaining DNA samples as concentrate as possible afterpurification, which will be diluted before HRM assay, overcomesinhibitors carryover problems present in a low quality DNA. TheHRM assay for ryr1 described here offers clear advantages overthe PCR-RFLP methodology published by Fujii et al. (1991).Moreover, HRM requires less optimization than similar systemsbased on TaqMan and fluorescence resonance energy transfer(FRET) probes. Compared to these methods HRM is a simplerand more cost-effective way to characterize multiple samples.Identification of human ryr1 SNPs using HRM has been alsodescribed by Grievink and Stowell (2007). In conclusion, the useof the HRM assay for porcine ryr1 genotyping considerablyreduces hands-on-time and costs. In addition, it is a closed assaysystem requiring no post-PCR processing and results arecomparable to conventional methods.

P080

DNA methylation biomarkers for noninvasive detectionof prostate cancer

Kristina Daniunaite1, Feliksas Jankevicius2,3, Juozas Lazutka1,Sonata Jarmalaite1

1Faculty of Nature Sci., Vilnius University, Lithuania; 2Faculty ofMedicine, Vilnius University, Lithuania; 3Vilnius University HospitalSantariskiu Clinics, Vilnius, Lithuania

Introduction. Prostate cancer (PCa) is the second most prevalentmalignancy of males characterized by high mortality rates. PCacan be effectively treated if it is diagnosed in its early stages,when the tumor is still confined to the prostate. Recently, aberrantDNA methylation in promoters of tumor suppressor genes hasbeen proposed as one of the most important events in prostatecarcinogenesis. It occurs at early stages of tumor developmentand can be detected by noninvasive means, including analysis ofurine sediments from PCa patients. Methods. For the developmentof noninvasive molecular biomarkers of PCa aberrant promotermethylation was analyzed in DNA from 102 samples of urinesediment from previously untreated cases of biopsy-proven earlyor medium stage PCa and 5 cases of benign prostatic hyperplasia(BPH). Quantitative methylation-specific PCR (QMSP) was usedfor sensitive detection of methylated DNA in catheterized urinespecimens. Genes GSTP1 (glutathione S-transferase pi 1),RASSF1 (Ras association (RalGDS/AF-6) domain family member1), and RARB (retinoic acid receptor beta), proven to be frequentlyhypermethylated in prostate cancer, were chosen for this analysis.The level of promoter methylation for particular gene wasevaluated by calculating percentage of methylated reference(PMR) using ACTB as endogenous control. Results. At least oneof the three genes was hypermethylated in urine sediments in 97of 102 PCa cases (95%), and 33 of 102 (32%) samples werepositive for methylation of at least two genes. RASSF1 wasmethylated in 94% (93 of 99), RARB – in 31% (32 of 102), andGSTP1 – in 14% (14 of 97) of the specimens. The average PMRfor positive cases was 40%, 5%, and 5% for RASSF1, RARB, andGSTP1, respectively. High level of methylation (PMR >50%) wasdetected in 35 of 93 (38%) cases for RASSF1, while PMR valuefor RARB and GSTP1 reached only 31% and 17%, respectively.Hypermethylation at relatively lower levels was also detected inurine sediments from BPH patients – average PMR was 20%,<1%, <1% for RASSF1, RARB, and GSTP1, respectively.Conclusions. Preliminary results of our study show high sensitivityof particular DNA methylation biomarkers for early andnoninvasive detection of prostate cancer.

P081

Does DNA methylation of the Estrogen Receptor Alphagene contribute to transcriptional regulation in thebovine endometrium?

Rainer W. Fürst, Heinrich H.D. Meyer, Susanne E. Ulbrich

Physiology Weihenstephan, Technische Universität München,Germany

Among the reproductive tissues, the endometrium undergoesfunctional changes during the estrous cycle which are regulatedby oestradiol-17β (E2) via the estrogen receptor alpha (ESR1).We asked whether differential methylation of an intragenic regionof this receptor plays an important role in its transcriptionalregulation during both estrous cycle and early pregnancy inbovine. Therefore, we monitored the mRNA expression of ESR1and investigated associated global and local DNA methylationpatterns from heifers slaughtered at estrus or 18 days after estrus(day 18 control), or during early pregnancy (day 18 - pregnant)(n=6 each). In addition, white blood cells from cyclic heifers wereanalyzed (n=6). Global DNA methylation was analyzed by theluminometric methylation assay (LUMA). Using high resolutionmelt (HRM) analysis, a specific area of the intron 1 regioncomposed of 16 CpG sites was amplified and subsequentlyanalyzed by pyrosequencing granting single CpG-site resolution.The endometrium was only weakly methylated (6 % averagemethylation). Neither HRM nor pyrosequencing of individual CpGsites revealed a significant difference between the analyzedgroups of endometrial samples. In contrast, leucocytes with onlyminute amounts of ESR1 transcripts showed a higher degree ofglobal methylation (83 % versus 76 % average methylation inendometrium, respectively) and also a significantly higher


methylation over the analyzed CpG sites (31% averagemethylation). In conclusion, DNA methylation of the analyzedregion of endometrial ESR1 seems not to be underlying thetranscriptional regulation during early pregnancy or cycle, althoughit might contribute to appropriate tissue-specific expression.

P082

Microarray gene expression and mtDNA deletionanalysis in laser dissected human muscle fibers.

Matthias Elstner1, Konrad Olszewski1, Christoph Laub1, AnneAverdam1, Lena Bruns1, Andreas Bender1, Holger Prokisch2,Thomas Klopstock1

1Ludwig-Maximilians University Munich, Germany; 2HelmholtzZentrum Munich, Germany

Background: Mitochondrial DNA (mtDNA) deletions causeprogressive external ophthalmoplegia (PEO), Kearns-Sayresyndrome (KSS), and Pearson syndrome. Single size deletions(SD) mostly occur sporadically in the germline, whereas mutationsof the nuclear-encoded mitochondrial replication machinery showmendelian transmission and cause multiple deletions (MD) ofvarious lengths. The phenotypic spectrum of mtDNA deletionsyndromes is highly variable, ranging from mild myopathy to thefatal multisystem KSS. This heterogeneity is considered to dependon the inter- and intraindividual variance of tissue deletion load, aswell as nuclear disease modifiers and environmental factors.Methods: We analyzed gene expression patterns of isolatedmuscle fibers from nine patients with PEO (5 female / 4 male, 6SD / 3 MD) using laser capture microdissection (LCM). Byexploiting the characteristic mosaic pattern of muscle tissue withcytochrome oxidase (COX)-positive and -negative fibers, weanalyzed deletion load and gene expression patterns in fibers withand without relevant OXPHOS dysfunction. Following RNAisolation and in vitro transcription, we determined whole genomeexpression patterns (Illumina®, WG6-V3) and examinedexpression changes using pathway analysis (Ingenuity®). Results:Deletion load reached a mean of 34% in COX-positive cells and60% in COX negative fibers (p=0.002). Microarray analysisidentified 138 significantly regulated genes (Benjamini & Hochbergfalse discovery rate 1%). Network analysis classified molecules asinvolved in regulation of DNA replication, recombination andrepair. Furthermore, top canonical pathways include folic acidsynthesis, protein ubiquitination and autophagy. Discussion: Byusing frozen human muscle biopsies and LCM, this methodologymay best approximate ‘in vivo’ transcriptome changes inmitochondrial deletion syndromes. By sampling fibers with normaland disrupted OXPHOS function from the same individual,confounding variables, such as age, sex, deletion type,heterogeneity and disease progression are abolished. We discussthe significance of our findings in the light of published data and itsrelevance regarding the understanding of disease mechanismsand therapy. Since deletions occur age-dependent in muscle andneurons, this data might also be of relevance for theunderstanding of sarcopenia and neurodegenerative disorders.

P083

From traditional RAPD-PCR to next generation HRMassisted real-time RAPD PCR

Irem Uzonur, Zeynep Katmer, Esma Ozsoy, Fatih Koyuncu,Tugba Senel

Fatih University, Turkey

Traditional random amplified polymorphic DNA (RAPD) techniqueis based on the amplification of DNA fragments using a shortarbitrary primer that anneals multiple locations on the genomicDNA. This is followed by separation of amplified fragments basedon their sizes using gel electrophoresis. Samples are identified bycomparing the DNA bands of the end-point fingerprints, which areexpected to be consistent for the same primer, DNA andexperimental conditions used. The real-time PCR system is basedon the detection and quantitation of a fluorescent reporter. Thissignal increases in direct proportion to the amount of PCR productin a reaction. By recording the amount of fluorescence emission ateach cycle, it is possible to monitor the PCR reaction duringexponential phase where the first significant increase in theamount of PCR product correlates to the initial amount of targettemplate. In addition, fluorescence data can be collected directlyfrom a real-time PCR instrument avoiding the drawbacks of end-

point detection. DNA melting analysis uses a double-strandedDNA fluorescent dye to detect single nucleotide polymorphisms(SNPs) and to perform mutation scanning following thepolymerase chain reaction (PCR) for DNA amplification. HighResolution Melt or HRM analysis is a powerful technique for thedetection of mutations, polymorphisms and epigeneticcomparisons through differences in melting plots of amplicons bymeasuring the melting temperature of amplicons in real time,using a fluorescent DNA-binding dye. In this study, a novel RAPD-PCR based method will be discussed to detect various levels ofgenetic variation exploiting the advantages of various next-generation qPCR applications such as new dye Technologies andHRM analysis.

P084

High Resolution Melting with Unlabelled Probeapproach for the analysis of Short Sequence TandemRepeats loci to sub-type Mycobacterium avium subsp.paratuberculosis

Matteo Ricchi, Gianluca Barbieri, Norma Arrigoni

IZSLER, National Centre for Paratuberculosis, Piacenza, Italy

Mycobacterium avium subsp. paratuberculosis (Map) is thecausative agent of paratuberculosis in ruminants and otherspecies. Many methods have been proposed to sub-type Map:multiplex PCR for IS900 integration loci, IS900 restriction fragmentlength polymorphism, amplified fragment length polymorphism,Pulsed Field Gel Electrophoresis and genotyping microarrays (1,2). However, at the moment, methods based on the amplificationof Micro- and Mini- satellites tandem-repeats loci are consideredthe emerging techniques for Map typing (1). In this regard, ShortSequence Tandem Repeats loci (SSR1, SSR2, SSR8 and SSR9)showed the highest discriminatory power; however, to identifythese loci, the direct sequencing of the amplicons is required. Ouraim was developing alternative methods to the sequencing. Wefocused on SSR8, which is constituted by triplets ranging fromthree to six repetitions. To identify each allele, we developed amethod based on an asymmetric qPCR, followed by High-Resolution Melting (HRM) analysis with unlabelled probes. Inorder to improve the efficiency of asymmetric PCR reaction, wedesigned primers accordingly to the Linear-After-The-Exponential(LATE) PCR strategy, while to avoid any elongation during theamplification, the unlabelled probe was blocked adding C6-aminogroup at its 3’ end. All PCR reactions were carried out on StepOnePlus system (Applied Biosystems, Milan, Italy) in a final volume of25 ml using LCGreen® Plus+ Melting Dye (Idaho technology Inc,Salt Lake City, USA) and Right-Taq (EuroClone, Pero, Italy). HMRanalysis was carried out according to Palais (3), without ROXnormalisation and smoothing the graphs with running averagemethod. For the development of the technique we used three Mapstrains containing, respectively, three, four and five repetitions; forthe allele with six repetitions, due to its rarity, we synthesised anartificial amplicon. HMR analysis showed two melting domains foreach sample: one relative to the amplicon omoduplex (DNAdouble strand) and another to the heteroduplex DNA single strand/probe. The omoduplex domain did not allow any differentiationbetween the various alleles, while the heteroduplex domainshowed almost 3 °C between each allele, permitting an unbiasedidentification. These data were confirmed analysing 40 Map fieldstrains. The method herein proposed is robust, reproducible, costeffective and faster than direct sequencing and could be usefullyapplied to resolve locus SSR8.

1.Motiwala AS, Li L, Kapur V, Sreevatsan S., Current understanding of thegenetic diversity of Mycobacterium avium subsp. paratuberculosis. 2006.Microbes Infect. 8, 1406-18.

2.Pribylova R, Kralik P, Pavlik I., 2009. Oligonucleotide microarraytechnology and its application to Mycobacterium avium subsp.paratuberculosis research: a review. Mol Biotechnol. 42, 30-40.

3.Palais R and Wittwer CT. 2009. Mathematical algorithms for high-resolution DNA melting analysis. Methods Enzymol.454:323-43.


RNAi: microRNA – siRNA Applications

P085

Titer dependent cytotoxicity of viral vectors measuredin real time via ECIS-technology

Jakob Müller1, Christian Thirion2, Pfaffl Michael W.1

1Technische Universität München, Germany; 2Friedrich-Baur-Institute, Klinikum der LMU, Department of Neurology, Germany

Recombinant viral vectors are widespread tools for transfer ofgenetic material in various modern biotechnological applicationslike functional studies, vaccine development, gene therapy or RNAinterference. However the practical handling often bears crucialproblems. An accurate and reproducible titer assignmentrepresents the basic step for most downstream applications ofviral vectors not only considering precise MOI adjustment. Asnecessary scaffold for the studies described in this work wedeveloped a qPCR based approach for viral particlemeasurement. Proximate a fundamental vector originated problemconcerning physiological effects is that the appliance of viralvectors can be attended by toxic effects on the individual cellculture model or tissue. To determine the individually critical viraldose we utilize Electric Cell-substrate Impedance Sensing (ECIS)to reveal toxic effects leading to cell death. It was first introducedin cell culture by Giaever and Keese and can be utilized forexamination of cell growth, cell motility, cell barrier function, invitro toxicology and is even applied in cancer research. With ECIStechnology the impedance change of a current flow through thecell culture medium in an array plate is measured in a non-invasive manner. The device visualizes effects on cellular levellike cell attachment, cell-cell contacts or proliferation. Here wedescribe the potential of this online measurement technique in anin vitro model using the porcine ileal ephithelial cell line IPI-2I incombination with an adenoviral transfection vector (Ad5 derivate).With this approach we can show a clear dose-depending toxiceffect. The amount of applied virus correlates with the level of celldeath. So this assay offers the possibility to discriminate theminimal non toxic dose of the individual transfection method.

P086

Specific and sensitive quantitative RT-PCR of miRNAswith DNA primers.

Susanna Cirera1, Ingrid Balcells2, Peter K. Busk3

1Genetics and Bioinformatics, University of Copenhagen, LIFE,Denmark; 2Unitat de Ciència Animal, University Autonoma ofBarcelona, Spain; 3Section for Sustainable Biotech, AalborgUniversity, Denmark

MicroRNAs are important regulators of gene expression and it isof great interest to quantitatively determine their expression levelin different biological settings. Here, we describe a PCR methodfor quantification of microRNAs based on a single reversetranscription reaction for all microRNAs combined with real-timePCR with two, microRNA-specific DNA primers. Primer annealingtemperatures were optimized by adding a DNA tail to the primersand could be designed with a success rate of 94 %. The methodwas able to quantify synthetic templates over eight orders ofmagnitude and readily discriminated between microRNAs withsingle nucleotide differences. Importantly, PCR with DNA primersyielded significantly higher amplification efficiencies of biologicalsamples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation thatlocked nucleic acid interferes with efficient amplification of shorttemplates. The higher amplification efficiency of DNA primerstranslates into higher sensitivity and precision in microRNAquantification. In conclusion, miR-specific quantitative RT-PCRwith DNA primers is a highly specific, sensitive and accuratemethod for microRNA quantification.

P087

Silencing of a Superoxide Dismutase in Phaedoncochleariae (Coleoptera: Chrysomelidae) by usingRNAi.

René Roberto Bodemann, Wilhelm Boland, Antje Burse

Max Planck Institute for Chemical Ecology, Germany

RNA interference (RNAi) is a well conserved mechanism oftranslation-inhibition in virtually all eucaryotic organisms. It givesthe opportunity to down regulate a single protein by using itsnucleotide sequence for synthesis of the interference-triggeringdouble stranded RNA (dsRNA) and observe effects of theabsence in vivo. Our model organism for checking the ways RNAicould probably work in chrysomelid beetles, is P. cochleariae. Asa member of the subfamily Chrysomelinae he exhibits nine pairsof dorsal accessory glands from which it can excrete droplets ofdefense secretion in cases of disturbance. The deterrents areeither sequestrated and modified plant glucosides, de novoproduced iridoids or derive from a mixture of both. The transportproteins and enzymes which make these three systems work arethe future targets of our RNAi. I started by silencing thesuperoxide dismutase (SOD) that has very high expression levelin the glands which makes it easy to observe. The enzyme,transported into the defense secretion, catalyses the reaction ofoxygen radicals, which are byproducts of deterrent-synthesis, tohydrogen peroxide. Different amounts of dsRNA corresponding toSOD sequence, have been injected in different life stages ofP.cochleariae and the expression-level of this SOD was analyzedby qPCR. Up to now I found out, that injections into middle instarslarvae lead to a 90 to 99% reduction of SOD mRNA levelcompared to control-groups which had been injected with thesame amount of not effecting GFP-dsRNA. Injections into pupaeor adults lead to similar effects. Interistingly following generationswas not affected in comparable dimensions. The next steps will beto analyze the correlation of qPCR results with proteinconcentrations and the search for an increased RNAi-effect inoffspring’s of pupae and adults. Furthermore siRNA experimentswill be done and an alternative delivery method for the dsRNA willbe investigated.

P088

Broad based profiling ~1150 microRNA designed inversion 16 of miRBase in a single experiment usingthe SmartChip System from WaferGen Biosystems

Tony Dodge, alan chang, Harita Veereshlingam, SangeethaAnandakrishnan, Sherry Wei, Roy Bohenzky

WaferGen Biosystems, United States of America

The SmartChip Real-Time System from WaferGen Biosystems isa platform for performing massively parallel real-time PCR assaysfor multiple applications, including gene expression studies. Thissystem combines the benefits of real-time PCR, includingsensitivity, precision and dynamic range, with the low cost persample and high throughput profiling power of hybridizationarrays. The system consists of three components; a SmartChip,comprising 5184 nanowells preprogrammed with gene-specificreaction content, a SmartChip Nanodispenser for applying sampleand reaction mix to the SmartChips, and a SmartChip Cycler forperforming and collecting data from the real-time PCR assays.Using cDNA as an input for this system, one can generatethousands of data points which can comprise a thorough portraitof relative gene expression in a sample. The mirnome has beengaining increasing focus as a target population of RNA inbiomarker profiling. The discovery tools available for profilinginclude labor intensive or limited accuracy methods in nextgeneration sequencing and hybridization arrays. Quantitative PCRhas been used extensively for profiling 100’s of microRNA on afew samples, but these methods can both be costly and provideonly singlicate data points, arguable only a qualitative tool formicroRNA profiling. Here we present a cost effective, broadlydefined and quantitative microRNA profiling tool in the SmartChipHuman microRNA Panel (version 2). A new ligation based methodis combined in a SYBR® Green quantitative PCR method. Over1150 microRNA assays have been designed to miRBase v16,allowing for a cost effective quantitative profiling tool with eachassay in quadruplicate per panel. We will describe the newchemistry, its performance with respect to sensitivity andspecificity, and representative data in control tissues.


P089

ChIP-qPCR and qbasePLUS jointly identify a MYCN-activated miRNA cluster in cancer

Barbara D'haene1, Pieter Mestdagh2, Daniel Muth3, FrankWesterman3, Frank Speleman2, Jo Vandesompele1,2

1Biogazelle, Zwijnaarden, Belgium; 2Center for Medical Genetics,Ghent University, Ghent, Belgium; 3Department of TumourGenetics, German Cancer Center, Heidelberg, Germany

Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) isvery well suited to assess and quantify direct binding of specificregulatory proteins to genomic DNA sequences. Unfortunately,data-normalization and accurate quantification appear to be amajor challenge for many users. Here, we demonstrate that ChIP-qPCR in combination with state-of-the-art real-time PCR data-analysis software enables convenient and reliable quantification.We applied ChIP-qPCR to assess binding of transcription factorMYCN to miRNA cluster 17-92, to a positive control target, MDM2,and to a negative control target region. ChIP-qPCR wasperformed in two MYCN-overexpressing neuroblastoma cell lines(IMR5 and WAC2) using SYBR Green I detection chemistry in a384-well plate and signals were normalized based on the averageabundance of three non-specific genomic regions in the ChIPsamples using the qbasePLUS multiple reference genenormalization technology. Fold enrichment was calculated relativeto the input sample (non-precipitated) and compared to that of afourth non-specific region (negative control target). Using thisapproach we were able to demonstrate strong MYCN-binding tothe positive control and the miR-17-92 cluster. In keeping with this,the expression level of the miR-17-92 cluster is substantiallyincreased in primary neuroblastoma tumor samples, in which theMYCN gene is amplified and overexpressed.

The results confirm the power of ChIP-qPCR in combination withthe data-analysis software qbasePLUS to study gene regulation.

P090

LNA-based miRNA qPCR facilitates the discriminationof different stages in cancer progression in atransgenic mouse model

Kim B Barken1, Ditte Andreasen1, Rolf Søkilde1, JanStenvang2, Niels Tolstrup1, Thomas Litman3, Boye S Nielsen1,Peter Mouritzen1

1Exiqon, Denmark; 2Copenhagen University, LIFE, Denmark;3Litman Consulting, Denmark

miRNAs are small RNAs (21-23nt) which function as post-transcriptional regulators of gene expression, possibly responsiblefor regulation of more than one third of the cellular transcriptome.Numerous studies provide evidence for a causal link betweenmiRNA dysregulation and several disease states, including humancancers. We have developed the miRCURY LNA™ Universal RTmiRNA qPCR platform to facilitate sensitive and accurate miRNAexpression profiling in both human, mouse and rat. Here we haveused the new Pick&Mix product where a custom selection ofassays is supplied in ready to use PCR plates. We have appliedthe miRCURY LNA™ Universal RT miRNA PCR platform to studyprogression of breast cancer in a genetically modified mousemodel and show that the expression pattern of miRNAs clearlyprovides a means to stage the cancer progression. In the study, agroup of wild type mice was compared with a group of polyomamiddle T antigen (PymT) transgenic mice. This mouse modelmimics the progression of human breast cancer and can be usedas an experimental model to investigate the mechanisms involvedin the process of metastasis. Samples were taken at differentprogression stages of the tumor and a selection of 18 highly and

differentially expressed miRNAs were identified by miRNA profilingon miRCURY LNA™ arrays. The expression of the 18 miRNAswas investigated by miRCURY LNA™ Universal RT microRNAPCR during three different progression stages of the cancer:Adenomas (early), early carcinomas (intermediate) andadenocarcinomas (late). Principal component analysis of theexpression of the 18 miRNAs clearly shows clustering of the earlytumor stage together with the wild type whereas both theintermediate and the late stage clearly clusters separate from thewild type. The results further confirm that the LNA-based miRNAqPCR serve as an ideal platform for disease biomarkeridentification.

Late submissions:

P091

qPCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2 (AAV2) ITR-Sequences

Aurnhammer, Christine; Haase, Maren; Muether, Nadine;Häusl, Martin; Huber, Ingrid; Nitschko, Hans; Busch, Ulrich;Sing, Andreas; Erhardt, Anja; Baiker, Armin

Organization(s): Bavarian Health and Food Safety Authority(LGL), Germany

Background: Viral vectors based on adeno-associated virusserotype 2 (AAV2) constitute promising tools in human genetherapy. The inverted terminal repeats (ITRs) within the viralgenome are the only cis-acting viral elements required forfunctional AAV2 vector generation and constitute the lowestcommon denominator of all AAV2-based vectors. However, so far,no PCR-based method for the detection and quantification ofAAV2-ITRs could be established due to their extensive secondaryhairpin structure. Current PCR-based methods are thereforepredominantly targeting vector-encoded transgenes or regulatoryelements.Methods: We established an AAV2-ITR sequence-specificquantitative PCR (qPCR) method. Primers and BBQ-labeled probeare located within a particular subregion of the ITR sequence andhave been designed to detect wild type AAV2 and AAV2-basedvectors.Results: This method is suitable for the evidence of both, single-stranded (genomic) DNA derived from AAV2 vector particles anddouble-stranded DNA derived from producer plasmids. The lineardynamic range could be shown for 102 to 107 copies and thedetection limit determined as 50 copies. A practical approach forthe analysis of putative cross reactivities against closely relatedAAV serotypes utilizing synthetic oligo nucleotides revealed somecross reactivity against orthologous sequences of AAV1, 3, 6 and7 but not against AAV4, 5, 8 and 9. For AAV vector productionadenovirus type 5 (Ad5) is often used in terms of its helper virusproperties. Therefore, we further investigated the specificity of ourqPCR method using Ad5-DNA and could prove the method toresult in no detectable cross reactivity with Ad5.Conclusion: This method comprises the first qPCR systemfacilitating the detection and quantification of AAV2 -ITRsequences. Since this method can be applied for all AAV2-basedvectors in a “one for all”-based manner, it will significantly simplifyAAV2 vector genome titrations in the future.


List of participants

Afif Abdel NourLaSalle Beauvais19, rue Pierre Waguet60000 Beauvais [email protected]

Elisabeth AberlTUM Z I E LWeihenstephaner Berg 385354 Freising [email protected]

Ivana AdamsQIAGENQiagen Str. 140721 Hilden [email protected]

Amalia Adler-BeutgenFujifilm Europe GmbHHeesenstr. 3140549 Düsseldorf [email protected]

Raza AhmedAgilent TechnologiesLSCA, Lakeside, Cheadle RoyalBusiness ParkSK8 3GR Stockport United [email protected]

Peter AignerInvetechIndustriestraße 258604 Volketswil [email protected]

Javier AlbaBio-Rad LaboratoriesGarrotxa 10-12, 1 A108820 El Prat [email protected]

Andreas AlbersRoche Diagnostics GmbH - DAMP 6164DAMP 616482377 Penzberg [email protected]

Ahmed AlharbiTaibah UniversityP.O. Box 876621492 Jeddah Saudi [email protected]

Christian AltenhoferTUMUrzenweg 264121 Altenfelden [email protected]

Ditte AndreasenExiqonSkelstedet 162950 Vedbaek [email protected]

Elisabeth Lill AndreassenArcticZymes ASSykehusveien 239294 Tromsø [email protected]

Birgit AnwaldBeckman Coulter Genomics GmbHAm Neuland 182347 Bernried [email protected]

Hans AttigQIAGENQiagenstraße 140724 Hilden [email protected]

Herbert AuerIRB BarcelonaBaldiri Reixac 1008028 Barcelona [email protected]

Madhu AugustineLife Technologies1149 Chess Drive94404 Foster City United [email protected]

Christine AurnhammerBavarian Health and Food SafetyAuthority (LGL)Veterinärstr. 285764 Oberschleißheim [email protected]

Jean-Christophe AvarreInstitut de Recherche pour leDéveloppementIRD-GAMET, BP 509534196 Montpellier cedex 05 [email protected]

Ali AyadiHabib Bourguiba Hospital: Sfax - TunisiaLaboratory of parasitology andmycology:Habib Bourguiba Hospital3029 Sfax [email protected]

Beatrix BahleRoche Diagnostics GmbHNonnewald 282377 Penzberg [email protected]

Stephan BählerQIAGEN Instruments AGGarstligweg 8CH-8634 Hombrechtikon [email protected]

Kim B BarkenExiqonBygstubben 92950 Vedbaek [email protected]

Ross Thomas BarnardThe University of Queenslandbuilding 68, room 3194072 Brisbane [email protected]

Christine BaumhauerDaiichi Sankyo Europe GmbHLochhamerstr. 29 RG82152 Martinsried [email protected]

Ingrid BayerHistologie Labor BayerBelgradstr. 880796 München [email protected]

Marc P. BealBiosearch Technologies81 Digital Drive94949 Novato United [email protected]

Hubert BecherAmplifaRickenbacher Strasse 10788131 Lindau [email protected]

Ingrid BechlerRoche Diagnostics GmbHRoche Diagnostics GmbH DAMP 616482377 Penzberg [email protected]

Christiane BeckerTUMLiesel-Beckmann-Strasse 685354 Freising [email protected]

Beatriz BellosilloHospital del MarPasseig Marítim 25-2908003 Barcelona [email protected]

Stefan BentinkExosome Diagnostics GmbHAm Klopferspitz 19a82152 Martinsried [email protected]

Bajram BerishaTUM WeihenstephanHittostr. 485354 Freising [email protected]

Tim BerschneiderRoche Diagnostics GmbHNonnenwald 282377 Penzberg [email protected]

Nadia BertazzonCRA-Centro di Ricerca per la ViticolturaViale XXVIII aprile, 2631015 Conegliano [email protected]

Holger BerthelBioline GmbHIm Biotechnologie Park TGZ 214943 Luckenwalde [email protected]

Arnaud BEURDELEYROCHE DIAGNOSTICS FRANCELab Network - Biologie Moléculaire38240 MEYLAN [email protected]

Daniela Bischof BoeschMicrosynth AGSchützenstrasse 159436 Balgach [email protected]

Francisco BizouarnBio-Rad Laboratories Inc2000 Alfred Nobel Drive94547 Hercules United [email protected]

Lotte BjerrumDanish Technological InstituteKongsvang allé 298000 Aarhus C [email protected]


Silvia BobenstetterLMULiebigstr.3885354 Freising [email protected]

Marie-Dominique BockSanofi-Aventis1, avenue Pierre Brossolette91385 CHILLY-MAZARIN [email protected]

René R. BodemannMax Planck Institute for ChemicalEcologyHans-Knoell-Str.8D-07745 Jena [email protected]

Harry BoeltzFluidigm EuropeParnassustoren1076 AZ Amsterdam [email protected]

Manja BöhmeBiotype Diagnostic GmbHMoritzburger Weg 6701109 Dresden [email protected]

Elena BorzovaUEA1 Cross LaneNR3 1BU Norwich United [email protected]

Hanna BragdeLänssjukhuset RyhovBuilding E3 Level 455185 Jönköping [email protected]

Maksim BratchikovBiosta Ltd.S.Zukausko 18-11708234 Vilnius [email protected]

Sabine BrauerIMGM Laboratories GmbHLochhamer Str. 2982152 Martinsried [email protected]

Bertram BrenigUniversity of GoettingenBurckhardtweg 237077 Göttingen [email protected]

Irena BrinkmannInstitute of Transfusion Medicine andImmunologyLudolf-Krehl Strasse 13-1768167 Mannheim [email protected]

Kai-Uwe Brodersen-van AalstIntegrated DNA TechnologiesInterleuvenlaan 12AB-3001 Leuven [email protected]

Melanie BroszatUniklinik FreiburgBreisacherstr. 115 b79106 Freiburg [email protected]

Nicolas BRUNETROCHE DIAGNOSTICS FRANCE2, avenue du Vercors38240 MEYLAN [email protected]

Florian BuckelMAB Discovery GmbHForstenriederstr. 8-1482061 Neuried [email protected]

Antje BurseMax Planck Institute for ChemicalEcologyHans-Knoell-Str.8D-07745 Jena [email protected]

Stephen Andrew BustinBarts and the London School of MedicineAcademic SurgeryE1 1BB London United [email protected]

Silvia CalatroniA.O. Treviglio-Caravaggiovia Folla di Sotto 1127100 Pavia [email protected]

Fabrizio CarlettiInmi Lazzaro SpallanzaniVia Nicola Laurantoni 5400149 Rome [email protected]

ANNA CASTELLÓUNIVERSITAT AUTONOMA DEBARCELONAFACULTAT DE VETERINARIA08193 BELLATERRA [email protected]

Paolo CatarsiIRCCS Policlinico San MatteoFoundation, Piazzale Golgi27100 Pavia [email protected]

Inge CellerTU München WeihenstephanWeihenstephaner Berg 385350 Freising [email protected]

Vladimir CelmareRoche Diagnostics Romania3-5 Piata Presei Libere, City GateBuilding, South Tower, Floors:4A,5,6, 1stDistrict Bucharest020335 Bucharest [email protected]

Urska CepinNational Institute of BiologyVecna pot 111SI-1000 Ljubljana [email protected]

Alexander M ChagovetzUniversity of Utah2315 East Sheridan Rd84108 Salt lake City United [email protected]

Caifu ChenLife Technologies850 Lincoln Centre Dr.94404 Foster City United [email protected]

Mark ChilcottBio-Rad2000 Alfred Nobel Drive94547 Hercules United [email protected]

Lily ChiuNational University Hospital5 Lower Kent Ridge Road119074 singapore [email protected]

Lai-on ChuDiagCor Bioscience Inc. Ltd.28/F, Tower A, Billion Centre,Kowloon Hong [email protected]

Lindsay ChungBIOKÉPlesmanlaan 1d2333BZ Leiden [email protected]

Susanna CireraUniversity of Copenhagen, LIFEGroennegaardsvej 3,1870 Frederiksberg [email protected]

Philippe CLAIRUNIVERSITE MONTPELLIER 2Campus Triolet34095 MONTPELLIER Cedex 5 [email protected]

Sabine CLEOPHAXBIOCORTECH8 rue Croix de Jarry75013 PARIS [email protected]

Aron CohenAzure PCR59A Brent StreetNW4 2EA London United [email protected]

Michelle CollinsBio-Rad Laboratories2000 Alfred Nobel Dr94547 Hercules United [email protected]

Ron M. Cook, Ph.D.Biosearch Technologies81 Digital Drive94949 Novato United [email protected]

Muriel CRAYNESTEUROGENTECrue du Bois Saint Jean 54102 Seraing [email protected]

Valentina Dall'OlioCogentechvia Adamello, 1620139 Milan [email protected]

Benedicte DanisUCB Pharma S.A.Chemin du Foriest1420 Braine-l'Alleud [email protected]


Kristina DaniunaiteVilnius UniversityM. K. Ciurlionio 21/27LT-03101 Vilnius [email protected]

Katrin DanowskiTU MünchenGeneral-von-Stein-Str. 8a85356 Freising [email protected]

Philip DayUniversity of ManchesterManchester Interdisciplinary BiocentreM1 7DN Manchester United [email protected]

Inky de BaereBiocartisAnwerpsesteenweg 2372350 Vosselaar [email protected]

Sabine DelannoyAgence Nationale de Sécurité Sanitaire(ANSES)23 avenue du général de Gaulle94706 Maisons-Alfort [email protected]

Dirk DemuthSigma-Aldrich Company Ltd.84 Burnage LaneM19 2WL Manchester United [email protected]

Holger DensowBiometra GmbHRudolf-Wissell-Straße 30D-37079 Göttingen [email protected]

Stefaan DerveauxWaferGen Biosystems7400 Paseo Padre Parkway94555 Fremont United [email protected]

François-Xavier Desforgesgenewavele Dorian b275011 Paris [email protected]

Jan DetmersChimera Biotec GmbHEmil Figge Str. 1044227 Dortmund [email protected]

Barbara D'haeneBiogazelleTechnologiepark 39052 Zwijnaarde [email protected]

Jeroen DijkstraRadboud University Nijmegen MedicalCentrePO Box 91016500 HB Nijmegen [email protected]

Thu Hien DoKatholieke Universiteit LeuvenKasteelpark Arenberg 13, bus 24553001 Leuven [email protected]

Zuzana DobsakovaCytogenetic Laboratory BrnoVeveri 39602 00 Brno Czech [email protected]

Stefanie DommelTU MünchenMühlenweg 111185354 Freising [email protected]

Maxime Lilian DoomsLaSalle BeauvaisStagiaire DISC - LaSalle Beauvais60000 Beauvais [email protected]

Roya DoroudiLife TechnologiesFrankfurther Str 129b64293 Darmstadt [email protected]

Brigitte DötterböckTU MünchenWeihenstephaner Berg 385350 Freising [email protected]

Mike DysonWaferGen Biosystems7400 Paseo Padre Parkway9455 Fremont United [email protected]

Christoph EckertMetabionLena-Christ-Str. 4482152 Planegg-Martinsried [email protected]

Franck EdouardBio-Rad3 boulevard Poincare92430 Marnes La Coquette [email protected]

Marlene EggertInstitut für HumangenetikGoethestraße 2980798 München [email protected]

Morten EldeArcticZymes AsSykehusveien 239294 Tromso [email protected]

Matthias ElstnerKlinikum GroßhadernMarchioninistr. 1581377 Munich [email protected]

Fons ElstrodtBIOKÉPlesmanlaan 1d2333BZ Leiden [email protected]

Markus EpeAffymetrixFürstenrieder Straße 27381377 München [email protected]

Maxime EugeneEUROGENTECRue du Bois Saint Jean 54102 Seraing [email protected]

Patrick FachFrench Agency for Food, Environmentaland Occupational Health (ANSES)23, avenue du général de Gaulle94706 Maisons-Alfort [email protected]

Aurore FalcozROCHE DIAGNOSTICS FRANCELab Network - Biologie Moléculaire38240 MEYLAN [email protected]

Netta FatalThermo ScientificKeilaranta 16A02150 Espoo [email protected]

Richard FeketeLife Technologies2130 Woodward St.78740 Austin United [email protected]

Eva FelderSanitätsakademie der BundeswehrNeuherbergstr.1180937 München [email protected]

Jovita Fernandez-PineroCentro de Investigación en SanidadAnimal (CISA-INIA)Ctra Algete a El Casar, s.n28130 Valdeolmos, Madrid [email protected]

Anna FerrerCenter for Genomic Regulation - CRGDoctor Aiguader, 808003 Barcelona [email protected]

LUISA FILIPPINCRA-VIT Centre for Research inViticultureviale XXVIII Aprile 2631015 CONEGLIANO [email protected]

Barbara FitzkyLife TechnologiesFrankfurter Str, 129B64293 Darmstadt [email protected]

Krzysztof FlisikowskiTechnical University of Munich, WZWLiesel-Beckmannstr. 185354 Freising [email protected]

Christine FochtmannTU MünchenWeihenstephaner Berg 385350 Freising [email protected]

Choy Len FongLife TechnologiesBlk 33, #05-03739256 Singapore [email protected]


FRANCESCA FONTANASILICON BIOSYSTEMS SPAVIA DEI LAPIDARI 1240129 BOLOGNA [email protected]

David KennardAzure PCR59A Brent StreetNW4 2EA London United [email protected]

Amin ForootanMultid Analyses ABOdinsgatan 2841103 Göteborg [email protected]

Roderic Alexander FueerstIT-IS Life Science LtdBldg 1000, Unit 1201 & 1202None Cork [email protected]

Andreas FunkeIDT Biologika GmbHAm Pharmapark06861 Dessau-Rosslau [email protected]

Rainer W. FürstTUMWeihenstephaner Berg 385354 Freising [email protected]

Hendrik FussMolecular Machines & IndustriesBreslauer Strasse 285386 Eching [email protected]

Mirco GeislerSigma AldrichEschenstr.582024 Taufkirchen [email protected]

Katharina GellrichPhysiology WeihenstephanAmpertal 1385777 Fahrenzhausen [email protected]

Lars GerdesBayerisches Landesamt für Gesundheitund Lebensmittelsicherheit (LGL)Veterinästr. 2D-85764 Oberschleißheim [email protected]

Matthew GibbsMars PetcareWaltham Centre for Pet NutritionLE14 4RT Melton Mowbray [email protected]

Alfons GierlDekanat der Fakultät WZWAlte Akademie 885354 Freising-Weihenstephan [email protected]

Christian GiersdorffPensionCapital GmbHKantstraße 280807 München [email protected]

Kelly GilesTechnology Networks LimitedBull Lane Industrial EstateCO10 0FD SUDBURY United [email protected]

Yadvinder GillMARS, WALTHAM CENTRE FOR PETNUTRITION,LE14 4RT LEICESTERSHIRE [email protected]

Winfried GirgQiagen GmbHQiagenstr. 140724 Hilden [email protected]

Florian GlaserEvotec AGSchnackenburgallee 11422525 Hamburg [email protected]

Viktoria GlunkBioEPS/TUMGaymannstr. 185354 Freising [email protected]

Irene GörzerMedical University of ViennaKinderspitalgasse 151095 Vienna [email protected]

Brigitte GramsamerInstMikroBioBwNeuherbergstraße 1180937 Munich [email protected]

Ursula GreplRoche Diagnostics GmbHNonnenwald 282377 Penzberg [email protected]

Elena GrigorenkoLife Technologies, Inc12 Gill Street Suite 400001801 Woburn United [email protected]

Diana GrochovaCytogenetic Laboratory BrnoVeveri 39602 00 Brno Czech [email protected]

Anna E GroebnerTechnische Universität MünchenWeihenstephaner Berg 385354 Freising [email protected]

Josef GrossTUMLiesel-Beckmann-Strasse 685354 Freising [email protected]

Cornelia GroßeUniversity Halle-WittenbergKurt-Mothes-Str. 306120 Halle [email protected]

Helga GruberBavarian Health and Food SafetyAuthorityVeterinärstr. 285764 Oberschleißheim [email protected]

Patrick GuertlerBavarian Health and Food SafetyAuthorityVeterinärstr. 285764 Oberschleißheim [email protected]

Michele GuesciniUniversity of UrbinoVia I Maggetti, 2661029 Urbino [email protected]

Marina GUILLETROCHE DIAGNOSTICS FRANCELab Network - Biologie Moléculaire38240 MEYLAN [email protected]

Sandra GUILLOUDROCHE DIAGNOSTICS FRANCELab Network - Biologie Moléculaire38240 MEYLAN [email protected]

Stephen GunstreamIntegrated DNA Technologies1710 Commercial Park52241 Coralville United [email protected]

Mandana Haack-SørensenRigshospitaletJuliane Mariesvej 20, 93022100 Copenhagen [email protected]

Heli HaakanaThermo Fisher ScientificRatastie 201620 Vantaa [email protected]

Gerd HaberhausenRoche Diagnostics GmbHNonnenwald 282377 Penzberg [email protected]

Knut HamannLifetechnologiesTheodor-Strom-Str. 1523863 Bargfeld-Stegen [email protected]

Matthias HanczarukBundeswehr Institute of MicrobiologyNeuherbergstr. 1180937 München [email protected]

Rita HärteisRoche Diagnostics GmbHNonnenwald 283277 Penzberg [email protected]

Luise HartmannTU MuenchenEhrenpreisstrasse 986899 Landsberg [email protected]


Karl H. HasensteinUniv. Louisiana Lafayette300 E. St. Mary Blvd70504-2451 Lafayette United [email protected]

Andrea HeckerMedical Faculty Mannheim, HeidelbergUniversity, Ludolf-Krehl-Strasse 13-1768167 Mannheim [email protected]

Torben HelledieExiqonSkelstedet 162950 Vedbaek [email protected]

Jan HellemansBiogazelleTechnologiepark 39052 Ghent [email protected]

Katharina HellerZIEL PhD-Kolleg "Epigenetik"Gregor-Mendel-Str. 285350 Freising-Weihenstephan [email protected]

Kaisa HelminenThermo ScientificKeilaranta 16A02150 Espoo [email protected]

Jana HemmerlingPhD Kolleg "Nutritional adaptation andepigenetic mechanisms"Gregor-Mendel-Str. 285350 Freising-Weihenstephan [email protected]

Anna HengerBiolytix AGBenkenstr. 2544108 Witterswil [email protected]

Neli HevirFaculty of MedicineVrazov trg 21000 Ljubljana [email protected]

Mirela HilaPediatric Hospital Sibiu9-9A Dimitrie pompeiu Street, building2A, ground floor020335 Bucharest [email protected]

Alison HillsGSTS Pathology, Guys and St ThomasNHS Foundation TrustCytogenetics DepartmentSE1 9RT London United [email protected]

Chris HingleyPrimerdesign LtdMillbrook Technology CampusSO15 0DJ Southampton United [email protected]

Kassie HiraniBiolineUnit 16 The Edge Business CentreNW2 6EW London United [email protected]

Akihiro HiranoRIKEN (The Institute of Physical andChemical Research, Japan)2-1 Hirosawa351-0198 Wako-city [email protected]

Hans-Joachim HoeltkeRoche Diagnostics GmbHRoche Diagnostics GmbHD-82377 Penzberg [email protected]

Hans HoenickaInstitute of Forest GeneticsSieker Landstr. 222927 Großhansdorf [email protected]

Gerhard HoferQIAGENQIAGEN Str.140724 Hilden [email protected]

Michael HoffmannRoche DiagnosticsNonnenwald 282377 Penzberg [email protected]

Ingrid HoffmannRoche Diagnostics GmbHNonnenwald 282377 Penzberg [email protected]

Janin HofmannIpal GmbHBundesallee 17110715 Berlin [email protected]

Béatrice HOFMANNEUROGENTECRue du Bois Saint Jean4102 Seraing [email protected]

Lars Christian HofmannClontech / Takara BioGuardinistr. 9581375 München [email protected]

Karsten HöhnAnalytik Jena AG / Biometra GmbHKonrad-Zuse-Straße 107745 Jena [email protected]

Barbara HolzerMedical University of ViennaWaehringer Guertel 18-20, 5Q1220 Vienna [email protected]

Nikos HontzeasSigma Aldrich2909 Laclede Ave.63103 St. Louis United [email protected]

Michaela HoockAgilent TechnologiesLautengartenstrasse 64052 Basel [email protected]

Martin HoratRoche Diagnostics AGForreenstraße6343 Rotkreuz [email protected]

Martin HorlitzQIAGEN GmbHQIAGEN Strasse 140724 Hilden [email protected]

Ralf HorresGenXPro GmbHAltenhöferallee 360438 Frankfurt am Main [email protected]

Eric HouptUniversity of VirginiaUniversity of Virginia22947 Charlottesville United [email protected]

Ilona HromadnikovaThird Faculty of Medicine, CharlesUniversity PragueRuska 8710000 Prague 10 Czech [email protected]

Ingrid HuberBayerisches Landesamt für Gesundheitund LebensmittelsicherheitVeterinärstr. 285764 Oberschleißheim [email protected]

YOUG MIN HUHYONSEI UNIVERSITY250 seongsanno, seodaemun-gu120-749 seoul South [email protected]

André ImhofMolecular Machines & IndustriesFlughofstrasse 378152 Glattbrugg [email protected]

Peter JacobsLifetechnologiesSuikerkaai 11500 Halle [email protected]

Aurélie JAMINEUROGENTEC5 rue du Bois Saint Jean4102 Seraing [email protected]

Greta JohannessonTATAA BiocenterOdinsgatan 2841103 Gothenburg [email protected]

Martin JudexAgilent TechnologiesKurt-von-Unruh-Weg 684085 Langquaid [email protected]

Katrin JulingEurofins Medigenomix GmbHAnzinger Str. 7a85560 Ebersberg [email protected]


Dafni-Maria KagkliEuropean Commission-Joint ResearchCentrevia Enrico Fermi 274921027 Ispra [email protected]

Olev KahreSolis BioDyneRiia 185a, 51014 Tartu, Estonia51014 Tartu [email protected]

Sebastian KaiserLMU MünchenLudwigstr. 3380539 München [email protected]

Andreas KalchschmidGenetic DI (Europe) AGAm Mittleren Moos 4886167 Augsburg [email protected]

Niels KaldenhovenGenmab BVYalelaan 603584 CM Utrecht [email protected]

Gustav KarlbergIlluminaChesterford Research ParkCB10 1XL Saffron Walden, Essex [email protected]

Joachim Richard KarlsenDanish Medicines AgencyAxel Heides Gade 12300 Copenhagen S [email protected]

Kersti KaskSolis BioDyne OÜRiia 185 a50415 Tartu [email protected]

Ly KäsperSolis Biodyne OÜRiia 185a, 51014 Tartu, Estonia51014 Tartu [email protected]

Zeynep KatmerFatih UniversityBuyukçekmece34500 Istanbul [email protected]

Christine KaufmannTechnische Universität MünchenWeihenstephaner Steig 2385354 Freising [email protected]

Ian KavanaghThermo Fisher ScientificBlenheim RoadTW2 5NH Epsom United [email protected]

Linda Veronique KazandjianMAB Discovery GmbHForstenriederstr. 8-1482061 Neuried [email protected]

Andreas KeckAffymetrixFürstenrieder Straße 27381377 München [email protected]

Antje KernAffymetrixFürstenrieder Straße 27381377 München [email protected]

David Michael KerryLife Technologies5791 Van Allen Way92008 Carlsbad United [email protected]

HYE SEON KIMYONSEI UNIVERSITY250 seongsanno, seodaemun-gu120-749 seoul South [email protected]

Seong-Youl KimBioneer Corporation49-3 Munpyung-dong, Daeduck-ku306-220 Daejeon South [email protected]

Benedikt KirchnerGiggenhauser Straße 185354 Freising [email protected]

Christoph KirschMacherey-Nagel GmbH & Co.KGNeumann-Neander-Straße 652355 Düren [email protected]

Hannes KirzingerRocheFritz-Schlamppstr. 586932 Pürgen [email protected]

Jörg KleiberHochschule WeihenstephanHochschule Weihenstephan85350 Freising [email protected]

Manfred KliemRoche Diganostics Deutschland GmbHSandhofer Str. 11668305 Mannheim [email protected]

Heike KliemTUM, Weihenstephaner Berg 385354 Freising [email protected]

Thilo KluetschAgilentSindesldorfer Strasse 60e82377 Penzberg [email protected]

Kevin L KnudtsonUniversity of Iowa323 EMRB52242 Iowa City United [email protected]

Andreas KochBASF SESpeyerer Strasse 267117 Limburgerhof [email protected]

Minna Riikka KoivulaThermo Fisher Scientific/Finnzymes OyRatastie 201620 Vantaa [email protected]

Varaprasad KollaUniversity Hospital, BaselHebelstr204056 Basel [email protected]

Michal KonecnySt. Elizabeth Cancer Institute81250 Bratislava [email protected]

Regina KonradBavarian Health and Food SafetyAuthorityVeterinärstr. 285764 Oberschleißheim [email protected]

Mirjana KozulicQIAGEN Instruments AGGarstligweg 88634 Hombrechtikon [email protected]

Vanessa KraftTechnische Universität MünchenLange Point 13/10185354 Freising [email protected]

Stefan KraissRoche Diagnostics GmbHNonnenwald 282377 Penzberg [email protected]

Petr KralikVeterinary Research InstituteHudcova 7062100 Brno Czech [email protected]

Koos KranenborgBIOKÉPlesmanlaan 1d2333BZ Leiden [email protected]

Jette Dina KreibergNovo Nordisk A/SNovo Nordisk Park2760 Måløv [email protected]

Vlastimil KrivdaState Veterinary Institute PragueSidlistni 136/24165 03 Prague Czech [email protected]

Guido KruppFujifilm / AmpTec GmbHKoenigstrasse 4A22767 Hamburg [email protected]

Fabian KubatyIDT Biologika GmbHAm Pharmapark06861 Dessau-Rosslau [email protected]


Mikael KubistaTATAA BIOCENTEROdinsgatan 1841113 Gothenburg [email protected]

Gerrit KuhnLife TechnologiesBaarer Str 786300 Zug [email protected]

Jaakko Tuomas KurkelaThermofisherRatastie 2 (P.O. Box 100)01620 Vantaa [email protected]

Elise LabbeLaSalle Beauvais19, rue Pierre Waguet60000 beauvais [email protected]

Laura F. LalondeCanadian Food Inspection Agency116 Veterinary RoadS7N 2R3 Saskatoon [email protected]

Olfert LandtTIB MolbiolEresburgstraße 22-2312105 Berlin [email protected]

Oliver LatzEurofins MWG OperonAnzinger Str. 7a85560 Ebersberg [email protected]

Robert LechnerTU MünchenHofmark 1-382393 Iffeldorf [email protected]

Hong Kai LeeNational University Hospital5 Lower Kent Ridge Road119074 Singapore [email protected]

Steve LefeverUniversity GhentDe Pintelaan 1859000 Gent [email protected]

Thomas LegrandGlaxoSmithKline Biologicals20, rue Fleming, Parc de la Noire Epine1300 Wavre [email protected]

Ursula LeiserRoche Diagnostics Ltd.Forrenstrasse6343 Rotkreuz [email protected]

Clarisse LemaitreLaSalle Beauvais19, rue Pierre Waguet60000 Beauvais [email protected]

Thomas LiemRoche DiagnosticsHochrütistrasse 396005 Luzern [email protected]

Antoon LievensInstitute of Public HealthRue Juliette Wytsmanstraat 141050 Brussel [email protected]

Kristina LindTATAA BiocenterOdinsgatan 28411 03 Gothenburg [email protected]

Andrea LinkmeyerTUM - WeihenstephanEmil-Ramann Str. 2D-85350 Freising [email protected]

Andrea LinnemannQiagen GmbHQiagenstr. 140724 Hilden [email protected]

Ken LivakFluidigm Corporation7000 Shoreline Court, Suite 10094080 South San Francisco [email protected]

Yuk Ming Dennis LoThe Chinese University of Hong KongDepartment of Chemical PathologyHK Hong Kong [email protected]

Stéphane LOBBENSROCHE DIAGNOSTICS FRANCELab Network - Biologie Moléculaire38240 MEYLAN [email protected]

Robert LoeweGeneWake GmbHForstenrieder Str. 1082061 Neuried [email protected]

Sabine LohmannRoche Applied ScienceNonnenwald 282377 Penzberg [email protected]

Katrin LorenzAnalaytik Jena AG / Biometra GmbHKonrad-Zuse-Straße 107745 Jena [email protected]

RANIERO LORENZETTIISTITUTO ZOOPROFILATTICOSPERIMENTALE DELLE REGIONILAZIO E TOSCANAVIA APPIA NUOVA 141100178 ROMA [email protected]

Jacek LubelskiAMT BiopharmaMeibergdreef 611105BA Amsterdam [email protected]

Karsten LuenoLife TechnologiesFrankfurter Strasse 129 B64293 Darmstadt [email protected]

Marie-Louise LunnExiqonSkelstedet 16DK-2950 Vedbaek [email protected]

Soumya MadhavanFriedrich-Schiller-University JenaNeugasse 2507743 Jena [email protected]

Rodrigo ManjarinMichigan State University1290 Anthony Hall48823 East Lansing United [email protected]

Christian MannThermo ScientificIm Steingrund 4-663303 Dreieich [email protected]

UGO MARCHESIISTITUTO ZOOPROFILATTICOSPERIMENTALE DELLE REGIONILAZIO E TOSCANAVIA APPIA NUOVA 141100178 ROMA [email protected]

Mercedes Marín-AguileraHospital Clínic of Barcelona-FundaciónClínicVillarroel 17008036 Barcelona [email protected]

Jan MarkusSt. Elizabeth Cancer InstituteHeydukova 1081250 Bratislava [email protected]

Eva MartinezHipraAvda. La Selva13517170 Amer [email protected]

LUZ MARTINEZ AVILESHOSPITAL DEL MARPASSEIG MARITIM08003 BARCELONA [email protected]

MARIOS MATARAGASAgricultural University of AthensIera Odos 75GR-11855 Athens [email protected]

Irene MaumneeKIC1855 W Taylor St60612 Chicago United [email protected]

Ralf MauritzRoche Diagnostics GmbHNonnenwald 283277 Penzberg [email protected]


Irena Mavric-PleskoKmetijski Institut SlovenijeHacquetova ulica 17SI-1000 Ljubljana [email protected]

Dietmar MayerIDT Biologika GmbHAm Pharmapark06861 Dessau Roßlau [email protected]

Nicole MayländerBavarian Nordic GmbHFraunhoferstrasse 1382152 Martinsried [email protected]

Frank McCaughanMedical Research CouncilMRC Laboratory of Molecular BiologyCB1 3AX Cambridge United [email protected]

Rena McCloryIllumina9885 Towne Centre Drive92121 San Diego United [email protected]

Johannes Peter MeierBioEPSLudwig-Thoma-Str.2384307 Eggenfelden [email protected]

Julia MeiserInstitute for Experimental and ClinicalPharmacology and ToxicologyKirrbergerstrasse66421 Homburg [email protected]

Jose Melo-CristinoFaculdade de Medicina da Universidadede LisboaAv Prof Egas Moniz1649-028 Lisboa [email protected]

Lourdes MengualHospital Clinic-Fundació ClínicVillarroel, 17008036 Barcelona [email protected]

ANNA MERCADEUniversitat Autonoma de BarcelonaFACULTAT DE VETERINARIA.O8193 BELLATERRA [email protected]

Steffen MergemeierCONGEN Biotechnologie GmbHRobert-Rössle-Str.1013125 Berlin [email protected]

Ssilvia MeschiInmi Lazzaro SpallanzaniVia Francesco Carrara 1655100 Lucca [email protected]

Swanhild MeyerTechnische Universität MünchenWeihenstephaner Berg 385350 Freising [email protected]

Heinrich HD MeyerPhysiology - WeihenstephanWeihenstephaner Berg 385354 Freising [email protected]

Dagmar MichelBiomedBruckmannring 3285764 Oberchleißheim [email protected]

Gabriel MinarikGENETON Ltd.Cabanova 14841 02 Bratislava [email protected]

Axel Moehrle4titude Ltd.Sickingenstrasse 2610553 Berlin [email protected]

Peter MolkenthinInstMikroBioBwNeuherbergstraße 1180937 Munich [email protected]

Claire MonkPrimerdesign LtdMillbrook Technology CampusSO15 0DJ Southampton United [email protected]

Shila MortensenStatens Serum InstitutArtillerivej2300 Copenhagen [email protected]

Martin MoserUniversity of ZurichWinterthurerstrasse 1908057 Zürich [email protected]

Ilona MoßbruggerInstitut für Mikrobiologie der BundeswehrNeuherbergstraße 1180937 München [email protected]

Cinthia MoysésLife TechnologiesAv Itacira, 210504061002 São Paulp [email protected]

Jakob MüllerTechnische Universität MünchenItzling 685354 Freising [email protected]

Elke MüllerGenetic ID (Europe) AGAm Mittleren Moos 4886167 Augsburg [email protected]

Alexander NagyState Veterinary Institute PragueSídlištní 136/24165 03 Prague 6 Czech [email protected]

Maren NeefUniversity of TuebingenAuf der Morgenstelle 172074 Tübingen [email protected]

Marcus NeusserBio-RadHeidemannstrasse 16980939 München [email protected]

Anthony NewmanElsevierRadarweg 291043 NX Amsterdam [email protected]

Bart NijmeijerAMT BiopharmaMeibergdreef 611105BA Amsterdam [email protected]

Gerd NilsenArcticZymes ASSykehusveien 239294 Tromsø [email protected]

Andreas NitscheRobert Koch InstituteNordufer 2013353 Berlin [email protected]

Hans NitschkoMax von Pettenkofer-InstitutPettenkoferstr. 9a80336 München [email protected]

Anna Krestine NoergaardDanish Technological Institute.Kongsvang Allé 29 Build. 48000 Aarhus [email protected]

Mikkel NoerholmExosome Diagnostics, GmbHAm Klopferspitz 19a82152 Martinsried [email protected]

Marika NymanTUMAM HOCHANGER 285350 FREISING [email protected]

Sören OcvirkBioEPSVöttinger Straße 34c85354 Freising [email protected]

Ralph OehlmannIMGM Laboratories GmbHLochhamer Str. 2982152 Martinsried [email protected]

Jakob ØrtvigAH DiagnosticsRunetoften 188210 Aarhus V [email protected]


Michael PacherSunGene GmbHCorrensstr. 306466 Gatersleben [email protected]

Shiyang PanThe first affiliated Hospital of NanjingMedical University300, Guangzhou Road, Nanjing, JiangsuProvince210029 Nanjing [email protected]

Renato PanebiancoUniversity Urbino - BiomolecularVia I Maggetti, 26/2 (Loc Sasso) 61029Urbino - Italy61029 Urbino [email protected]

Walter PaperEppendorf Vertrieb Deutschland GmbHJohann-Schmidbauer-Str. 494351 Feldkirchen [email protected]

Dario PapiTIB MolbiolEresburgstraße 22-2312105 Berlin [email protected]

SPIROS PARAMITHIOTISAgricultural University of AthensIera Odos 75GR-11855 Athens [email protected]

Viresh PatelBio-Rad Laboratories2000 Alfred Nobel Drive94547 Hercules United [email protected]

Filip PattynGhent UniversityDe Pintelaan 1859000 Gent [email protected]

Natasha PaulTriLink BioTechnologies, Inc.9955 Mesa Rim Road92121 San Diego United [email protected]

Michael W PfafflTUMWeihenstephaner Berg 385354 Freising [email protected]

Sylvia PfafflBioEPS GmbHLise-Meitner-Strasse 3085354 Freising [email protected]

Matthias PfeifferEurofins MWG OperonAnzinger Str. 7a85560 Ebersberg [email protected]

Henrik PfundhellerExiqonSkelstedet 162950 Vedbaek [email protected]

Paolo PiazzaWellcome Trust Centre for HumanGeneticsRoosevelt DriveOX3 7BN Oxford United [email protected]

Paul PickeringLife Technologies850 Lincoln Centre Drive, MS 44794404 Foster City United [email protected]

Janin PieritzLife TechnologiesGutenbergstr.10870197 Stuttgart [email protected]

Veronika PistekTU München, WeihenstephanWeihenstephaner Berg 385354 Freising [email protected]

Camille PlusquellecBIOCORTECH8 rue Croix de Jarry75013 Paris [email protected]

Michelle PlusquinHasselt UniversityCampus Diepenbeek3590 Diepenbeek [email protected]

Ingo PoleschakAgilent TechnologiesHauptstrasse 6285716 Unterschleissheim [email protected]

Kok-Siong PoonNational University Hospital5 Lower Kent Ridge Road119074 Singapore [email protected]

Sabina IurianPediatric Hospital Sibiu9-9A Dimitrie Pompeiu street, building2A, ground floor020335 Bucharest [email protected]

Derek PotterNanoString Technologies530 Fairview Avenue N98109 Seattle United [email protected]

Hervé PouzoullicLife Technologies25, Av de la Baltique B.P. 96Courtaboeuf91943 Villebon sur Yvette Cedex [email protected]

Rob PowellPrimerDesign LtdMillbrook Technology CampusSO150DJ Southampton United [email protected]

Bukkaraya, S PrakashNDRI KarnalNDRI132001 Karnal [email protected]

Sebastian PünzelerAdolf-Butenandt-Institut fürMolekularbiologieSchillerstraße 4480336 München [email protected]

Giorgia PuorroUniversità degli Studi di Napoli "FedericoII"Via Pansini 5, ed. 1780131 Naples [email protected]

Alexander RaczEurofins MWG OperonAnzinger Str. 7a85560 Ebersberg [email protected]

Eugen RaduEmergency University HospitalBucharest9-9A, Dimitrie Pompeiu Street, building2A, ground floor020335 Bucharest [email protected]

Arjun RajUniversity of Pennsylvania210 S. 33rd St.19104 Philadelphia United [email protected]

Ann RandolphUniversity of Michigan Health Service1150 W. Medical Center Dr.48109 Ann Arbor United [email protected]

Sarah DugdaleGeneSys LtdInnovation HouseGU16 7PL Camberley United [email protected]

Carolyn ReifsnyderAgilent Technologies, Inc.11011 N. Torrey Pines Rd.92037 La Jolla United [email protected]

Rita S. ReinRoche Applied ScienceNonnenwald 282377 Penzberg [email protected]

Martina ReiterBioEPS GmbHLise-Meitner-Strasse 3085354 Freising [email protected]

Volker ReuckTIB MolbiolEresburgstraße 22-2312105 Berlin [email protected]

Hyoung Seok RhoNovomics Meditech250 Seongsanno, Seodaemun-Gu120-752 Seoul South [email protected]


Matteo RicchiIZSLERVia Strada Faggiola 129027 Gariga di Podenzano [email protected]

Nicolas RICHARDROCHE DIAGNOSTICS FRANCELab Network - Biologie Moléculaire38240 MEYLAN [email protected]

Estelle RicheMerck Millipore1 rue J. MonodF-78280 Guyancourt [email protected]

Heidi RichterMetabionLena-Christ-Straße 4482152 Planegg- Martinsried [email protected]

Stefan RickenbachRoche Diagnostics AGz.H. Antonia Vögel6343 Rotkreuz [email protected]

Alain RICOLife Technologies25 avenue de la baltique91943 Courtaboeuf [email protected]

Irmgard RiedmaierTU MunichWeihenstephaner Berg 385354 Freising-Weihenstephan [email protected]

Mary RileyLonza191 Thomaston Street04841-2994 Rockland United [email protected]

Konstantin RizosGenetic ID (Europe) AGAm Mittleren Moos 4886167 Augsburg [email protected]

Yu-Hui RogersJ. Craig Venter Institute9704 Medical Center Drive20850 Rockville United [email protected]

Lorenz RoggoRoche Diagnostics (Switzerland) LtdIndustriestrasse 76343 Rotkreuz [email protected]

Sameer RohatgiIllumina9885 Towne Centre Drive92121 San Diego United [email protected]

Felix RollBiolegioLagelandse Weg 566545 CG Nijmegen [email protected]

Marianna RomzovaIBT ASCRVidenska 108314200 Prague 4 Czech [email protected]

Scott D. RoseIntegrated DNA Technologies1710 Commercial Park52241 Coralville United [email protected]

Rudi RossauPhilipsHigh tech campus 115656 AE Eindhoven [email protected]

Michael RosskopfQIAGEN GmbHQIAGEN Strasse 140724 Hilden [email protected]

Bernhard RothNovartis Vaccines & Diagnostics GmbHEmil-von Behring Str.7635041 Marburg [email protected]

Veronika RozehnalDaiichi-Sankyo EuropeLochhamer Str. 29a82152 München [email protected]

Jan M RuijterAcademic Medical Center, AmsterdamMeibergdreef 151105AZ Amsterdam [email protected]

Susan RümmlerOncoscreen GmbHLöbstedter Str. 9307749 Jena [email protected]

Matevž RuparNational Institute of BiologyVečna pot 1111000 Ljubljana [email protected]

Yvet NoordmanAMT BiopharmaMeibergdreef 611105BA Amsterdam [email protected]

Vendula RusnakovaInstiture of BiotechnologyVidenska 108314202 Prague Czech [email protected]

Ze'ev RussakAzure PCR59A Brent StreetNW4 2EA London United [email protected]

Thomas RygusLife TechnologiesFrühlingsweg 3a86859 Holzhausen [email protected]

Francesco SaccàUniversità degli Studi di Napoli "FedericoII"Via Pansini, 580127 Napoli [email protected]

Angela SachsenhauserTU MünchenWeihenstephaner Berg 385350 Freising [email protected]

Yamuna SahadevanMax Planck Institute for ChemicalEcologyEmma Heintz strasse 6a07745 Jena [email protected]

Roswitha SantnerLife TechnologiesOttenser Marktplatz 822765 Hamburg [email protected]

Ossian SarisThermo Fisher ScientificRatastie 2(P.O.Box 100)01620 Vantaa [email protected]

Mihir SarkarIndian Veterinary Research InstitutePhysiology & Climatology Division243122 Bareilly [email protected]

Christof Schaefauergenewavele dorian b275011 paris [email protected]

Dieter SchamsTUM WeihenstephanWeihenstephaner Berg 385354 Freising [email protected]

Kimberly Kay SchartlLONZA Biologics, GmbHNattermannallee 150829 Köln [email protected]

Barbara SchechingerIlluminaChesterford Research ParkCB10 1XL Saffron Walden, Essex [email protected]

Silvio K. ScheelQIAGEN GmbHKonrad-Peutinger-Straße 481373 München [email protected]

Pia ScheuBio-Rad LaboratoriesHeidemannstrasse 16480939 Muenchen [email protected]

Thomas Andreas SchildLife TechnologiesKantstr. 476137 Karlsruhe [email protected]


Michael SchleichertLambda GmbHGewerbepark 24261 Rainbach [email protected]

Melanie SchneiderOncoscreen GmbHLöbstedter Str. 9307749 Jena [email protected]

Alfred SchöllerLK Weinviertel MistelbachLiechtensteinstrasse 672130 Mistelbach [email protected]

Kati SchroederRobert Koch-InstitutNordufer 2013353 Berlin [email protected]

Rainer SchusterAmplifaRickenbacher Straße 10788131 Lindau [email protected]

Rachel ScottBio-Rad2000 Alfred Nobel Drive94547 Hercules United [email protected]

Daniela SebahLGLRingstrasse 2885764 Oberschleissheim [email protected]

Eva-Maria SedlmeierTechnische Universität MünchenGregor-Mendel-Straße 285350 Freising-Weihenstephan [email protected]

Bernhard SetzerBS-DiagnostikWaidplatzstrasse 879331 Teningen-Nimburg [email protected]

Amir Houshang ShemiraniDebrecen UniversityNagyerdei krt 984012 Debrecen [email protected]

Francois-Xavier SicotTakara Bio Europe2 avenue KennedyF-78100 Saint Germain en Laye [email protected]

Anna SiddleGeneSys Ltd.Innovation HouseGU167PL Camberley United [email protected]

Matthias W. SieberUniversity Hospital JenaErlanger Allee 10107743 Jena [email protected]

Franziska SiegelRheinische Friedrich-Wilhelms-Universität BonnSigmund-Freud-Str. 2553105 Bonn [email protected]

Tanja SiglTechnische Universitaet MuenchenVeitsmuellerweg 485354 Freising [email protected]

Carl-Johan SimolaLonzaStrandhaven 122665 Vallensbaek [email protected]

Davide SistiUniversity of UrbinoVia I Maggetti, 26/261029 Urbino [email protected]

Kerstin SkovgaardTechnical University of DenmarkBülowsvej 271790 Copenhagen [email protected]

Iva SlanaVeterinary Research InstituteHudcova 7062100 Brno Czech [email protected]

Michal SlanyVeterinary Research InstituteHudcova 7062100 Brno Czech [email protected]

Karen SmeetsHasselt UniversityAgoralaan, Building D3590 Diepenbeek [email protected]

Leila SmithFluidigm EuropeParnassustoren1076 AZ Amsterdam [email protected]

Jan SödermanRyhov County HospitalLänssjukhuset RyhovSE-55185 Jönköping [email protected]

Denny SonnemansIntervet International bvBuilding PP385830AA Boxmeer [email protected]

Ketil Bernt SorensenDanish Technological InstituteKongsvang Alle 298000 Aarhus C [email protected]

Diana SorgTechnische Universität MünchenWeihenstephanerberg 385354 Freising [email protected]

Virpi SorriThermo ScientificKeilaranta 16A02150 Espoo [email protected]

Ben SowersBiosearch Technologies81 Digital Drive94949 Novato United [email protected]

Mary SpanCYCLERtestRotscherweg 616374 XW Landgraaf [email protected]

Andrej-Nikolai SpiessUniversity Hospital Hamburg-EppendorfMartinistr. 5220246 hAMBURG [email protected]

Wolf D. SplettstoesserBundeswehr Institute of MicrobiologyNeuherbergstr. 1180937 Munich [email protected]

Stefan SpringerFH WeihenstephanSanddornweg 685375 Neufahrn [email protected]

Margrit StadlerFluidigm EuropeParnassustoren1076 AZ Amsterdam [email protected]

Anders StåhlbergUniversity of GothenburgCancer Center, University ofGothenburg, Box 42540530 Gothenburg [email protected]

Karin StangelandRoche Diagnostics Norge ASPb 66100607 Oslo [email protected]

Michael SteidleFujifilm Europe GmbHHessenstr. 3140549 Düsseldorf [email protected]

Julia SteigerBioline GmbHBiotechnologie Park TGZ 214943 Luckenwalde [email protected]

Tanja SteinLife TechnologiesFrankfurter Strasse 12964293 Darmstadt [email protected]

Oliver StephanLife TechnologiesFrankfurter Strasse 129b64293 Darmstadt [email protected]


Vilberto StocchiUniversity of UrbinoVia I Maggetti, 2661029 Urbino [email protected]

Martin Thomas StockLS für Zoologie WZW TUMHöhenweg 1387656 Untergermaringen [email protected]

Katharina StöckerIntervet Innovation GmbHZur Propstei55270 Schwabenheim [email protected]

Michael Richard StrakaIDEX Health & Science600 Park Court94929 Rohnert Park United [email protected]

Linda StrömbomTATAA BiocenterOdinsgatan 2841103 Göteborg [email protected]

Chanchal Sur ChowdhuryUniversity of BaselUniversity Hospital Basel, Department ofBiomedicine (ZLF)4031 BASEL [email protected]

David SvecTATAA Biocenter ABOdinsgatan42680 Gothenburg [email protected]

Tomas SzemesGenetonCabanova 1484102 Bratislava [email protected]

Monika Szyszka-NiagolovBiosystems Ltd.2, 6th September Str.1000 Sofia [email protected]

Jens-Eike TäubertTU MünchenMühlenweg 2285354 Freising [email protected]

Roxana TeisanuEcole Polytechnique Federale deLausanneAI 1112 (Bâtiment AI)CH-1015 Lausanne [email protected]

Gudrun TellmannRoche Diagnostics GmbHNonnenwald 282377 Penzberg [email protected]

Ales TichopadAcademy of Science of Czech RepublicVidenska 108314000 Prague Czech [email protected]

Jessica TiedkeUniversity of HamburgMartin-Luther King Platz 320144 Hamburg [email protected]

Laura TizzoniCogentechvia Adamello, 1620139 Milan [email protected]

Natasa ToplakOmega d.o.o.Dolinskova 81000 Ljubljana [email protected]

Kirsten UebelTU MünchenGregor-Mendel-Str. 285354 Freising-Weihenstephan [email protected]

Susanne E. UlbrichTechnische Universität MünchenWeihenstephaner Berg 385354 Freising [email protected]

Andreas UntergasserUniversity of HeidelbergSchlossstr 3464720 Michelstadt [email protected]

Irem UzonurFatih UniversityBuyukcekmece34500 Istanbul [email protected]

Agne ValinciuteState Research Institute of InnovativeMedical CenterBitininku str. 2B-2808310 Vilnius [email protected]

Laura ValinottoBuenos Aires Children's HospitalMoldes 2969 7A1429 Buenos Aires [email protected]

Koenraad van AckerBiocartisAntwerpsesteenweg 2372350 Vosselaar [email protected]

Pim van der AarDync B.V.Hortensiastraat 84818GM Breda [email protected]

Ronald van EijkLeiden University Medical CenterAlbinusdreef 22300RC Leiden [email protected]

Ruud van der SteenBiolegio BVLagelandse Weg 566545 CG Nijmegen [email protected]

Rob van MiltenburgRoche Diagnostics GmbHDAMP 616482377 Penzberg [email protected]

Jo VandesompeleGhent University & BiogazelleDe Pintelaan 1859000 Ghent [email protected]

Petra VasickovaVeterinary Research InstituteHudcova 7062100 Brno Czech [email protected]

David VAUDRYROCHE DIAGNOSTICS FRANCELab Network - Biologie Moléculaire38240 MEYLAN [email protected]

Markus VeitSigma AldrichEschenstr.582024 Taufkirchen [email protected]

Enrique ViturroTechnische Universität MünchenWeihenstephaner Berg 385354 Freising [email protected]

Hans von BesserEppendorfOberer Kühlenberg1497078 Würzburg [email protected]

Tobias WagnerLife TechnologiesDietramszeller Platz 781371 München [email protected]

Heiko WalchRocheNonnenwald 282377 Penzberg [email protected]

Rudolf WalserAmplifaHattnauerstrasse 1888142 Wasserburg [email protected]

Mandy WarnkeMolecular Machines & Industries AGFlughofstrasse 378152 Glattbrugg [email protected]

Barbara WeckerleinMetabion International AGLena-Christ-STr.4482152 Planegg-Martinsried [email protected]

Rosemarie WeikardLeibniz Institute for Farm Animal BiologyWilhelm-Stahl-Allee 218196 Dummerstorf [email protected]


Karin Tarp WendtTechnical University of DenmarkBülowsvej 271790 Copenhagen [email protected]

Jochen WettengelTechnische Universität MünchenGiggenhauserstr. 185354 Freising [email protected]

Jim WhiteNanoString Technologies21 Acrefield ParkL25 6JX Liverpool United [email protected]

Jim WicksPrimerdesign LtdMillbrook Technology CampusSO15 0DJ Southampton United [email protected]

Steffi WiedemannChristian-Albrechts-Universität KielOlshausenstr.24098 Kiel [email protected]

Marina WiklanderSigma-AldrichSolkraftsvagen 14CSE-135 70 Stockholm [email protected]

Anja Mareen WildQIAGEN GmbHQiagenstr. 140724 Hilden [email protected]

Wilhelm WindischTUMLiesel-Beckmann-Strasse 685354 Freising [email protected]

Carsten WinterBiometra GmbHRudolf-Wissell-Straße 30D-37079 Göttingen [email protected]

Vital WohlgensingerMicrosynthSchützenstrasse 159436 Balgach [email protected]

Roman WölfelBundeswehr Institute of MicrobiologyNeuherbergstrasse 1180937 Munich [email protected]

Tine Yding WolffThe Danish Technological InstituteKongsvang Allé 298000 Aarhus [email protected]

Aaron WoodleyTechnology Networks LimitedBull Lane Industrial EstateCO10 0FD SUDBURY United [email protected]

Patricia Susan WynneInstitut for PathologySchönbeinstrasse 404031 Basel [email protected]

Michal WysockiTUMLiesel-Beckmann-Straße 185354 Freising [email protected]

Remziye YILMAZMiddle East Technical University06530 Ankara [email protected]

ByoungSu YoonKyonggi University8114, Dept. Life Science443-760 Suwon South [email protected]

Yulia ZabiyakaMechnikov Research Institute ofVaccines and Sera, RAMSFirst Dubrovskaya st., 15115088 Moscow [email protected]

Stephan ZellmeierBio-Rad Laboratories GmbHHeidemannstr. 16480939 München [email protected]

Martin ZevenbergenKeygene N.V.Agro Business Park 906708 PW Wageningen [email protected]

Philip ZimmermannETH ZürichUniversitätsstrasse 28092 Zurich [email protected]

Barbara ZschörnigJena Bioscience GmbHLoebstedter Str. 8007749 Jena [email protected]

© 2011 by Michael W. Pfaffl

qPCR 2011 Event5th International qPCR Symposium Industrial Exhibition & Application WorkshopsMolecular Diagnostics: from single-cells to Next Generation Sequencing

ISBN 9783000338403

Editor:Michael W. Pfaffl E-mail [email protected], Freising – Weihenstephan Tel ++49 8161 713511Weihenstephaner Berg 3 Fax ++49 8161 713539Technical University Munich (TUM) WWW www.gene-quantification.info

Documents

qPCR 2011 Event Proceedings 5th International qPCR ... · 16:00 Single Molecule RNA FISH: novel, simple, and accurate quantitative applications for gene expression analysis Arjun