qPCR & Digital PCR Congress · 2015-04-24 · separate biological variation from technical one. In this talk, I will address some of the key issues in breadth of application, sensitivity

Recent advances in digital PCR and a reduction in cost have enabled the barriers that once held up the adoption of the technology to beovercome. A high performance alternative to qPCR providing absolute quantification, improved sensitivity & specificity, higher precision andgreater robustness is now far more accessible. With a recent survey from Frost & Sullivan finding that 30% of non digital PCR users plan topurchase digital PCR in 2013 and with the gene amplification market predicted to grow to $1.9 billion by 2015, this conference provides a timelyopportunity to learn first-hand about dPCR whilst also keeping up to date with latest developments and strategies in qPCR.

Attracting 150 industry & academic experts working in areas such as molecular biology / diagnostics, gene expression, genomics, biomarkers,pathogen detection, GMO, mRNA, NGS, bioinformatics and data management, the conference will examine the latest strategies, developmentsand case studies using the technologies to further research in areas such as oncology, infectious diseases, vaccines, prenatal diagnosis,diagnostic & clinical applications, microbiology, food microbiology, plant / ecology genomics and other novel applications.

Unlike other meetings, the qPCR & digital PCR Congress will feature 40 international speakers drawn from academia and industry. This uniqueblending of academia and industry will provide you with the widest possible range of views on qPCR research and also on the developmentsin digital PCR, as this new technology becomes more widespread. The Congress has been structured to deliver insights into the latest researchthrough a series of commentaries, panel discussions, case-studies and poster-presentations featuring developments, not just in Humanresearch, but also Plant/Ecology, and Food research.

Confirmed Speakers includeINDUSTRYThierry Bonnevay, Microbiology PlatformHead, Sanofi Pasteur, France

Yiu-Lian Fong, Executive Director,Companion Diagnostic Development,Novartis Oncology, USA

Mao Mao, Research Fellow, OncologyResearch, Pfizer Worldwide Research andDevelopment, USA

Arndt Schmitz, Founding Scientist, GlobalBiomarker - Research Biobank, BayerPharma AG, Germany

ACADEMIAAfif Michel Abdel Nour, AssociateProfessor, Special Infectious Agents Unit,King Fahad Medical Research Center, KingAbdulaziz University, Jeddah, Saudi Arabia

Barbara Brezna, VÚP Food ResearchInstitute, Bratislava, Slovakia

Philip Day, Reader in QuantitativeAnalytical Genomics, Manchester University,Director of Quantitative MolecularMedicine and Principal Investigator at theManchester Interdisciplinary Biocentre, UK

Tanja Dreo, Department of Biotechnologyand Systems Biology, National Institute ofBiology, Slovenia

Ronald van Eijk, Laboratory Researcher,Department of Pathology, Leiden UniversityMedical Center, The Netherlands

Pierre Laurent-Puig, Director, ProfessorMolecular Basis of the Response toXenobiotics, Descartes University, France

David Rodríguez-Lázaro, AssistantProfessor of Microbiology, University ofBurgos, Spain

Michael Ryckelynck, ISIS, University ofStrasbourg, France

Anders Ståhlberg, Senior Scientist, Dept ofPathology, University of Gothenburg, Sweden

www.globalengage.co.uk/qpcr.html

qPCR & Digital PCR CongressDevelopments & Potential of qPCR & dPCR as a Tool

for Progressing Molecular Biology Research

September 9th – 10th 2013, Hilton Lyon, France

Keith Jerome, Professor and Head,Virology Division, Department ofLaboratory Medicine, University ofWashington, Associate Member, Vaccineand Infectious Disease Division, FredHutchinson Cancer Research Center, USA

Philippe Corbisier, Scientific / TechnicalProject Manager, Joint Research Center,Institute for Reference Materials andMeasurements (IRMM), Standards forInnovation and Sustainable Development(SID), European Commission, Belgium

Emiliano Toso, Molecular Biology Head,Merck Serono, Italy

gold sponsor sponsor media partners

qPCR & Digital PCR Congress – September 9th – 10th 2013

For more information please contact Nick Noakes, Marketing Director, Global Engage [email protected] +44 (0) 1865 849841

Day One Stream One

qPCR Strategies & Developments

• Choosing your qPCR system

• qPCR assay design & optimisation

• MIQE guidelines & standardisation

• Quality control of qPCR assays

• Sample preparation & quality control

• Single cell qPCR analysis

• qPCR uses in clinical trials

Day One Stream Two

Digital PCR Possibilities & Opportunities

• Introduction, benefits, future development of digital PCR

• Converting to digital PCR from qPCR

• Panel discussion: qPCR v digital PCR

• Verifying accuracy of digital PCR data

• Complimenting digital PCR with other technologies

• Microfluidic tools to compliment digital PCR

• NGS v digital PCR – advantages over NGS

• Single cell analysis

• Best practice guides to use

• Panel discussion: qPCR v digital PCR

Day Two Stream One

qPCR & Digital PCR Case Studies• Clinical / diagnostic applications

• Cancer - gene mutations etc.

• Infectious diseases

• Prenatal diagnosis

• Vaccines

• Molecular diagnostics

• Biomarkers / validation

• microRNA / ncRNA / siRNA applications

• Genetically modified organism quantification in food

• Plant / ecology genomics

• Panel discussion: Clinical laboratories as a market fordPCR platforms

Day Two Stream Two

Bioinformatics, Data Analysis &Management and Plant & Food Case Studies• Automated analysis of qPCR data

• Challenges and new solutions for data analysis

• Gene expression profiling – qPCR

• High resolution melt (HRM) analysis

• Plant and food case studies

• Pathogen detection

• GMO

Conference Agenda

qPCR

Digital PCR

Microarrays

NGS

Microfluidics

Single Cell Analysis

Nucleic Acids

Absolute Quantification

Gene Expression

Genotyping

Genomics

Genomic Profiling

Biomarkers

Molecular Diagnostics

Pharmacogenomics

Epigenetics

Assay Development

Western Blotting

Copy Number Variation

Rare Event Detection

DNA Methylation

Absolute Counting

SNP Genotyping

Mutation Detection

Bioinformatics

Biostatistics

Data Analysis

Validation

High Resolution Melting

Analytical R&D

mRNA / SiRNA

Microbiology

Molecular Biology

Oncology

Infectious Diseases

Diagnostics

Prenatal Diagnostics

Vaccines

Plant Genomics

Food Microbiology

Who should attendDelegates are pre-qualified dependent on seniority, budget, responsibility & are senior-level decision makersfrom academic & industry institutions, mainly in Europe but also worldwide & typically include, Professors, VPs,Directors, Managers, Heads, Scientists of or working in:

Agenda Day 1 – Monday September 9th 2013


08.00 – 08.50 Registration & Coffee

08.50 – 09.00 Global Engage Welcome Address & Chairman’s Opening Remarks

09.00 – 09.30 Characterization of Cells at the Single-Cell Level• The added value of performing gene expression profiling at single-cell level• Workflow for analyzing DNA, RNAs and proteins in the same single-cell• Applications of single-cell analysis: Identification and characterization of novel subpopulations.

CONFIRMED:Anders Ståhlberg, Associate Professor, Sahlgrenska Cancer Center, Dept. of Pathology, University of Gothenburg, Sweden

09.30 - 10.00 Solution Provider PresentationAdvanced Applications of Droplet Digital PCR Since its relatively recent introduction, the field of applications of digital PCR has rapidly expanded to more than justnucleic acid quantification. Some of the more innovative uses of the QX100 ddPCR system will be described in thispresentation, outlining the wide range of applications enabled by this technology.

CONFIRMED:Yann Youvenot, Staff Scientist, Bio-Rad Laboratories

Sponsored by:

qPCR Congress

10.00 – 10.05 Stream Chair

10.05 - 10.30 Single Cell Analysis of Leukemia Heterogeneity and Drug Response• Single cell measurements of DNA, mRNA and protein forbcr-abl was measured in K562 cells to produce a steadystate model of BCR.ABL protein abundance per mRNAmolecule.

• The model predicts the level of BCR.ABL proteinheterogeneity in CML cells and supports effectivetherapeutic strategies including the combinedapplication of imatinib.

• The necessity for employing single cell measurementsover bulk measurements are compared to determine ifany benefit is gained through generating models basedon data from single cells.

• Novel in-vivo sampling from single cells is discussed.

CONFIRMED:Philip Day, Reader in Quantitative Analytical Genomics,Manchester University, Director of Quantitative MolecularMedicine and Principal Investigator at the ManchesterInterdisciplinary Biocentre, UK

10.30 - 10.55 Single Cell and Single Molecule DNA Methylation Analysisof Multiple Genes Using qPCR • Epigenetic mechanisms play an important role in normalcellular processes as well as in diseases such as cancer.

• Samples of interest are in most cases a heterogeneousmixture of different cell populations, which could displaydifferent methylation profiles depending on the cell type.

• Very few methods are currently applicable to theanalysis of DNA methylation patterns in a single cell.

• We developed a method for the analysis of the DNAmethylation status of several genes or regions of interestin FACS sorted single cells by multiplex PCR and real-time duplex PCR.

• Detailed quantitative methylation as well as allele-specific methylation status information can be obtainedby subsequent pyrosequencing.

CONFIRMED:Jorg Tost, Head, Laboratory for Epigenetics andEnvironment (LEE), Centre National de Génotypage, CEA - Institut de Génomique, France

Digital PCR Congress

Stream Chair

Introduction, Benefits, Future Development of dPCR• dPCR versus qPCR • Overcoming qPCR limitations

CONFIRMED:Yiu-Lian Fong, Executive Director, Companion DiagnosticDevelopment, Novartis Oncology, USA

Copy Number Value Assignment of Certified ReferenceMaterials by Digital PCRDigital PCR allows to quantify DNA fragments without theuse of standard curves and is therefore a method ofchoice to quantify calibrators that are needed forquantitative PCR.Since the concentration is derived by dividing the copynumber estimated by the assay volume, the partition ordroplet volume and its uncertainty need to be taken intoaccount when measuring DNA concentration by digital PCR.The ERM-AD623 CRM has been certified to contain BCR-ABLtargets at different levels varying between 106 to 10 cp/µL. Those quantities determined by dPCR have been confirmedby droplet digital PCR when taking into account the realvolume of the generated droplets.

CONFIRMED:Philippe Corbisier, Scientific / Technical Project Manager,Joint Research Center, Institute for Reference Materialsand Measurements (IRMM), Standards for Innovationand Sustainable Development (SID), EuropeanCommission, Belgium



qPCR Congress

12.15 - 12.40 Single-Cell Quantitative RT-PCR: Technical and AnalyticalChallenges in Exploring HeterogeneityRecent technological advances have allowed the explorationof gene expression programmes at the level of individualcells and contributed to the notion that functionally identicalcells can have heterogeneous transcriptional compositionsin a manner that influences cell fate. In pursuing the roleof heterogeneity in stem and developmental cell systems,it is critical that (1) the technology is applicable to relevantprimary cells types, namely those of low extractable RNAcontent, and that (2) the methodology can reliablyseparate biological variation from technical one.In this talk, I will address some of the key issues inbreadth of application, sensitivity and reproducibility ofsingle-cell quantitative RT-PCR technology with a focuson the Fluidigm microfluidics platform. I will then discussa pipeline of analytical methods that systematicallyexplore global and gene-specific aspects of molecularheterogeneity, including inference of gene regulatorynetworks and modelling of cell fate decisions.

CONFIRMED:Cristina Pina, NHS - Blood and Transplant, Department ofHaematology, University of Cambridge

12.40 - 13.05 Single Cell Analysis of Microchimeric SamplesThe analysis of microchimeric cells longs for single cellanalysis under rare cell conditions. In this context I willshow data of sample pre-enrichment strategies such asimmunomagnetic beads enrichment and filtration andfurther talk about how to identify microchimeric cells bymeans of DNA-typing - a conditio sine qua non in rarecell analysis. In the last part I will discuss our currentefforts to implement qPCR and digital PCR strategies intoour workflow.

CONFIRMED:Thomas Kroneis, Head of Research Unit for Single CellAnalysis, Institute of Cell Biology, Histology &Embryology, Medical University Graz, Austria


Digital PCR Strategies in the Development and Analysisof Molecular Biomarkers for Personalized Medicine• Intra-tumoral heterogeneity should play a key role inthe future care of cancer patients

• Therefore the development of sensitive method todetect it is important

• Detection of low frequency subclones in colorectaltumor and its clinical implications

CONFIRMED:Pierre Laurent-Puig, Director, Professor Molecular Basisof the Response to Xenobiotics, Descartes University,France

ctDNA and Digital PCR • Tumor DNA why? Stratification marker for noveltherapeutics

• Tumor DNA as stratification marker / example: lung Caguidelines

• From a scientifically sound marker to a clinical tool:concept of biomarker project work in pharma industry

Our partnering concept, a triangle of mutual win winsbetween clinicians, PhDs in pharma, and CROs/biotechs• digital PCR, the concept and pros• digital PCR, overview of commercially availablesolutions

CONFIRMED:Arndt Schmitz, Founding Scientist, Global Biomarker -Research Biobank, Bayer Pharma AG, Germany

13.05 - 14.00 Lunch - One-to-One Meeting

10.55 - 11.45 Morning Refreshments Poster Presentations

11.45 - 12.15 Solution Provider PresentationNanoString Technology the Bridge to the Clinic for Nucleic Acid BiomarkersThe nCounter System from NanoString Technologies, uses direct single molecule imaging with molecular barcodes tomultiplex and detect up to 800 targets in a single reaction direct from total RNA, cell lysates, FFPE, LCM or single cellsamples. The output is digital, one molecule one count , with a typical five-log dynamic range with a 50ng input oftotal RNA. The assay technology captures nucleic acid targets through hybridization, providing a simple workflow,with minimal pipetting, enhancing the precision and reproducibility of peer-to-peer analysis.The system supports applications for gene expression analysis, copy number analysis, ChIP-String-epigenetic analysis,and miRNA profiling.The nCounter System has proven capability with FFPE samples, as a tool to profile gene signatures in clinically relevantsamples. With the potential to accelerate the development of companion diagnostics for disease profiling withincreased statistical power.

CONFIRMED:Jim White, Field Support Specialist, Nanostring Technologies



14.00 - 14.30 Solution Provider PresentationFor sponsorship opportunities please contact Nick Best at [email protected]

15.20 - 16.10 Afternoon Refreshments Poster Presentations

qPCR Congress

14.30 - 14.55 Applying the MIQE Guidelines to Clinical and Pre-ClinicaltrialsMinimum Information for the “Publication of qPCRexperiments’’ guidelines are targeted at gene expressionexperiments. To our knowledge they have not been appliedto qPCR assays carried out in the context of clinical trials. This report details the use of the MIQE qPCR app for iPhone(App Store, Apple) to assess the MIQE compliance of oneclinical and five pre-clinical trials. This resulted in the needto include 14 modifications that make the guidelines morerelevant for the assessment of this special type of application. We also discuss the need for flexibility, since while someparameters increase experimental quality, they also requiremore reagents and more time, which is not always feasiblein a clinical setting. The second part of my talk will be anupdate on the MIQE-qPCR app through numbers since 2011.

CONFIRMED:Afif Michel Abdel Nour, Associate Professor, SpecialInfectious Agents Unit – King Fahad Medical ResearchCenter, King Abdulaziz University, Jeddah, Saudi Arabia

14.55 - 15.20 qPCR validation of the IFN Signature Quenching byBAMINERCEPT in PhaseII Clinical TrialsA subset of patients with autoimmune diseases includingrheumatoid arthritis (RA) and lupus appear to be exposedcontinually to interferon (IFN) as evidenced by elevatedexpression of IFN induced genes in the cells in the blood.Detection of endogenous chromatin complexes by theinnate sensing machinery is the suspected driver for the IFN,but the exact mechanisms remain unknown. Rheumatoidarthritis DMARD and TNF incomplete responder patients intwo separate clinical studies were treated with baminercept,a decoy receptor blocker of the lymphotoxin-a/b/LIGHTaxis. RNA IFN signatures and blood counts were measuredand patients from a SLE registry served for comparison.Administration of baminercept led to a reduced RNA IFNsignature in the blood of patients with elevated baselinesignatures. Global RNA IFN signatures, myeloid-specificSIGLEC1 expression and protein levels of the IFN-induciblechemokine CXCL9 were decreased following 3 months oftreatment. Both RA and SLE patients with a high IFN signaturewere similarly lymphopenic and lymphocyte countsincreased following baminercept treatment of RA patients.

CONFIRMED:Jadwiga Bienkowska, Principal Investigator, PatientStratification and Translational Research, Biogen Idec, USA


DNA-Methylation Testing / Biomarker Validation UsingHigh Throughput qPCR• Methylation sensitive restriction enzyme (MSRE) coupledqPCR is an efficient tool for DNA methylation analysesand validation of candidate markers

• Strategy enables cfDNA testing • Lung cancer DNA methylation markers derived fromtissue based screening successfully validated inpatients’ plasma DNA

CONFIRMED:Andreas Weinhäusel, Health & EnvironmentDepartment, Molecular Medicine, AIT Austrian Instituteof Technology, Vienna, Austria

qPCR is an Important Tool in Next GenerationSequencing• QC and QT of Next Generation Sequencing Libraries• New methods for high throughput gene expressionanalysis

• NGS vs dPCR – Advantages of NGS

CONFIRMED:Hannah Haydon, Scientific Officer, Genomics CoreFacility, Cancer Research UK

17.30 Chairman’s Closing Remarks and End of Day 1

17.30 - 18.30 Drinks Reception

qPCR Congress

16.40 - 17.05 The Use and Usefulness of Amplification Curve Analysisin Quantitative PCR: Impact and PerformanceQuantitative real-time RT-PCR or qPCR is the preferredtechnique for the quantification of RNA. In PCR, theamplicon concentrations per cycle are given by Nc=N0.E^C,in which N0 and Nc are amplicon concentrations at thestart and after C cycles and E is the amplification efficiency.The number of cycles (Cq) needed to reach a fluorescencethreshold (Nq) is then used to calculate N0 (=Nq/E^Cq).Most qPCR analysis methods are based on re-arrangements,and simplifying assumptions, of this basic equation andthus require the estimation of Cq and E. Often dilutionseries are used to derive E but this procedure was shownto give variable results. The performance of methods toderive the PCR efficiency from the log-linear part ofindividual amplification curves were compared.

CONFIRMED:Jan Ruijter, Dept. Anatomy, Embryology & Physiology,Academic Medical Centre, University of Amsterdam, The Netherlands

17.05 - 17.30 Multi-Gene Expression Signatures for Prediction ofCancer Subtype-Specific TraitsToday public resources host an enormous amount ofinformation on the expression of genes in different typesof cancers, on the response of cancer cells to externalstimuli, or even about association with drug sensitivity.The results of such studies can be captured by thedevelopment of databases of multi-gene expressionsignatures. However, it is often unclear whether thepredicitvity of expression signatures is maintained in anew data set that may have been generated in a differentexperimental set-up. A framework will be presented thatis addressing the question how the "translateability" and"relevance" of a multi-gene assay in new data can beassessed - a core question during the development ofPCR-based multi-gene assays from genome-wideexpression results.

CONFIRMEDEike Staub, Computational Biologist for Oncogenomics,Merck Serono and Lecturer of Bioinformatics, HochschuleMannheim, Germany


qPCR, dPCR and NGS for Detecting Mutations in TumorTissues and PlasmaThe mutations in EGFR such as L858R and exon 19deletions can predict clinical response to the EGFRinhibitors gefitinib and erlotinib in lung cancer, whilemutations like T790M confer resistance to the two drugs.The assays for screening these mutations allow selectingappropriate therapies and monitoring resistance. Thistalk will focus on comparing three different technologies,qPCR, dPCR and NGS, for detecting mutations in FFPEtumor specimens and plasma.

CONFIRMED:Mao Mao, Research Fellow, Oncology Research, PfizerWorldwide Research and Development, USA

Utilizing PCR Based Techniques for Biomarker Discoveryand Clinical ImplementationGenome Wide Association Studies (GWAS) have deliveredlots of information about genetic associations to disease,but relatively few clinical tests. One reason for this is thatdiseases are complicated and diverse phenotypes. A potential solution to this complexity is to study“intermediate phenotypes” such as the host immuneresponse. We have therefore initiated a 1000 healthydonor population based study to try to determine geneticand environmental associations for immune responsevariance. A key component of this study is examining theinduced immune response to a range of medicallyrelevant stimuli such as bacteria, viruses, and cytokines.We are currently exploring different gene expressiontechnologies such as digital PCR, which will identifyreliable signals that can be associated with host geneticvariance. Finally one of the clinically most useful GWASresults to date was the identification of the IL28B SNP as apredictor for outcome in Hepatitis C patients. We havedeveloped a point of care SNP PCR test to improve HepatitisC patient management in developing country settings.

CONFIRMEDDarragh Duffy, Project Manager, Immunobiology ofDendritic Cells, Institut Pasteur



16.10 - 16.40 Panel Discussion: qPCR v Digital PCR• Quality controls and quality of the data that you could get from dPCR• Limitations of current dPCR• Interface between dPCR and absolute quantification by NGS protocols

CONFIRMED:Panel Chair - Yiu-Lian Fong, Executive Director, Companion Diagnostic Development, Novartis Oncology, USAJonathan Frampton, Diagnostics Product Manager, Horizon Discovery LtdKeith Jerome, Professor and Head, Virology Division, Department of Laboratory Medicine, University of Washington,Associate Member, Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, USAMao Mao, Research Fellow, Oncology Research, Pfizer Worldwide Research and Development, USAAnders Ståhlberg, Associate Professor, Sahlgrenska Cancer Center, Dept. of Pathology, University of Gothenburg, Sweden


Agenda Day 2 – Tuesday September 10th 2013

09.00 – 09.30 Keynote Address

Digital PCR in the Clinical LaboratoryDigital PCR has to date been a technology predominantly used in the research setting. Thus, clinical laboratoriesrepresent a largely untapped market for dPCR platforms and applications. In this talk, Dr. Jerome will review emerginguses of dPCR in clinical virology, and discuss the degree to which dPCR is likely to supplant current qPCR utilization.

CONFIRMED:Keith Jerome, Professor and Head, Virology Division, Department of Laboratory Medicine, University of Washington,Associate Member, Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, USA

09.30 - 10.00 Reliable qPCR Data Analysis Enables Relevant Conclusions of Gene ExpressionqPCR technology is widely recognized as a relevant methodology and gold-standard for the analysis of geneexpression. Nevertheless, like all other scientific experiments qPCR data analysis should also be subjected to arigorous analytical procedure. RealTime StatMiner addresses the various data analysis needs of the users and helpsthem to perform a critical analysis of their gene expression data. The ability to automatically detect outliers and filter low-expressed genes as well as alerting the user to problemswithin their data at various points forms the comprehensive quality control metrics that is fundamental to dataanalysis in RealTime StatMiner. Integrating algorithms such as GeNorm, NormFinder and Minimum Variance Median toselect the best endogenous genes, RealTime StatMiner offers the ability to compute DCt based on user instructions.Furthermore, the package allows the user to select specific statistical tests for their data analysis. We will show youhow to perform rapid and reliable qPCR statistical analysis independent of the scientific discipline in a user friendlyenvironment with interactive visualizations and publication ready reports.

CONFIRMED:Eduardo Gonzalez, Chief Strategy Officer, Integromics

Sponsored by:

10.30 - 11.10 Morning Refreshments

11.10 - 11.40 Solution Provider PresentationFor sponsorship opportunities please contact Nick Best at [email protected]

qPCR and dPCR Case Studies

10.00 – 10.05 Stream Chair

10.05 - 10.30 Use of qPCR Technology in Quality Control Laboratoryunder GMP Environment for the Control of Vaccines• General presentation of molecular biology methods in aquality control laboratory under cGMP environment forthe control of vaccine

• Specific use of qPCR methods for the control of quality ofcommercialized and under development vaccines:different examples of application

• Difficulties and advantages of utilization of qPCR basedmethods in Quality Control laboratories

CONFIRMED:Thierry Bonnevay, Microbiology Platform Head,Analytical Research and Development Department,Sanofi Pasteur, France

Plant & Food Case Studies

Stream Chair

The Impact of MIQE Guidelines in Plant Science CommunityCurrently in plant research, validated reliable RT-qPCRprotocols are still rare. The methods used are often not atall performed according to the MIQE-guidelines. Thenecessity of using multiple reference genes has becomeobvious in quite some cases, but assay-specific validationof these genes is often lacking. Also RNA quality control isa crucial bottleneck. Machines for capillary electrophoresisallow to determine RNA quality quite easily, but RIN or RQIvalues do not apply on plant material. The use of noRTsamples is another delicate point. In our experience, theappearance of samples of which the Cq-value of the noRTis within 5 units of the actual sample are common inmost experiments. Ignoring this information can lead toa severe overestimation of the gene expression in aspecific sample. These three problems will be discussedmore profoundly in view of the necessary application ofthe MIQE-guidelines in plant research.

CONFIRMED:Ellen De Keyser, Senior Researcher Molecular Genetics &Breeding, ILVO (Institute for Agricultural and FisheriesResearch) - Plant Sciences Unit - Applied Genetics &Breeding, Belgium

13.30 - 13.55 Let’s get Digital: Powerful Approaches for Quantificationof Mutant and Methylated Sequences with Low AllelicBurdens

• Accurate detection of low level mutant and methylatedalleles requires a digital approach

• Limiting dilution enables effective analysis of single orlow copy numbers of sequences

• New technological developments enable highly costeffective analysis of very high numbers of replicates

CONFIRMED:Alex Dobrovic, Principal Research Fellow and Head,Molecular Pathology Research and Development, PeterMacCallum Cancer Centre, Australia

13.55 - 14.20 Cancer Mutation Detection

CONFIRMED:Ronald van Eijk, Laboratory Researcher, Department of Pathology, Leiden University Medical Center, The Netherlands

A qRT-PCR Assay for the Specific Expression of all theGenes of the Mal d 1 Family in Apple (Malus Domestica)and Applications on Fruits and LleavesThe increasing number of sequenced genomes has led tothe discovery of extensive gene duplication and retentionin plants and thus, of a high number of gene families. Theaim of this study was to develop and validate an assay forthe specific characterization of all the members of a familyby using a qRT-PCR and SNPs between genes. Mal d 1gene family was selected as model and a comprehensive,sensitive and affordable assay for studying the specificexpression of all the 31 known Mal d 1 genes wasobtained. The first application of the assay on apple fruitsand leaves upon biotic stress showed a great variabilityin gene expression among the family providing newperspectives for research on plant breeding.

CONFIRMED:Giulia Pagliarani, University of Bologna

A Loop-Mediated Amplification (LAMP)-Based DetectionMethod for Salmonella spp. in Animal Feed• Brief introduction to LAMP• LAMP vs PCR?• Some challenges

CONFIRMED:Martin D’Agostino, Microbiologist, The Food andEnvironment Research Agency



12.30 - 13.30 Lunch - One-to-One Meeting


11.40 - 12.05 Design, Development, Validation and Implementation ofResidual DNA Quantification Method Based on qPCRMethod for Quality Control of Vaccines

• DNA quantification methods in general• development of an in house design qPCR method for thequantification of residual DNA from eukaryotic cells

• Validation of the methods and establishment ofreferences

• implementation, results and specification, validitycriteria, discussion

CONFIRMED:Olivier Vernay, Manager, Microbiology Platform, SanofiPasteur, France

12.05 - 12.30 Molecular Biology GMP Tools for Biotech Quality Control:Developments & Potential Applications for the Future• Molecular Biology perceptions and trends• Development of a successful GMP platform: lessonslearned

• qPCR & NGS technology revolutions

CONFIRMED:Emiliano Toso, Associate Director and BQC MolecularBiology Head, Merck Serono, Italy


qPCR and dPCR in Detection and Identification of PlantPathogens: Development, Accuracy and Speed• qPCR assay design for plant pathogen detection,validation and quality control in day-to-day testing

• non-linear modelling of limit of detection• Cq cut-off values – a valid approach or a bit of cheating?

CONFIRMED:Tanja Dreo, Researcher, Department of Biotechnology andSystems Biology, National Institute of Biology, Slovenia

The Assay Development, Validation and Application ofTaqman Quantification of Pathogens forEnvironmental/Grain Samples

CONFIRMED:Theo Van der Lee, Senior Scientist MolecularPhytopathology, Wageningen UR


14.20 - 14.45 Detection Of Low Frequency Alleles In Patient Samples ByDigital PCR• Highly sensitive and quantitative detection of tumormarkers in patient samples

• Detection of circulating tumoral DNA• Detection of low frequency subclones in tumor andclinical implications

CONFIRMED:Valerie Taly, Group Leader Translational Research andMicrofluidics, Universite Paris Descartes, France

14.45 - 15.10 Miniaturization of Molecular Biology Using Droplet-Based Microfluidics: Digitalization Beyond the PCRDigital PCR has revealed to be a powerful tool indiagnostics and genotyping applications, when used as astand-alone analytical method. In addition, when used ina proper format, the application scope of digital PCR canbe readily expanded by combining it with other molecularbiology techniques to create more complex digitalmethodologies. In this presentation I will show howrecent developments in microfluidics were used to set acompletely in vitro and digital procedure to performeddirected evolution of biomolecules in a high throughputregime with perfect a control on experimental conditions.

CONFIRMED:Michael Ryckelynck, ISIS, University of Strasbourg, France


Pre-PCR Sample Prep: Past Disappointments, PresentKnowledge, and Future HopesSample preparation is a difficult and filthy job, which is whymost scientists prefer to work on the detection step developingfancy analytical methods: Nano-sensors and probe techniquesare certainly more impressive and more prestigious to work with.But what is the use of high-tech device, when the murkydiagnostic sample “kills” the signal. The technology “cemetery”is full of large and micro-devices that have not survived thereal-life testing. This dilemma has caused manydisappointments in the past. That is why an increasing numberof labs are turning their focus on the sample prep step in orderto understand the impact of various ingredients and chemicalson subsequent molecular reactions. This is currently creatingmuch generic knowledge with substantial scientific interest.Besides food testing, research grants to projects on bio-terror,metagenomic-based geo-microbiology, genomic anthropology,molecular evolution, etc. is helping to develop better (andhopefully simpler) sample prep method that can resist the toughconditions of routine testing. Here of course the decisive elementis the cost: while human cancer diagnostics can pay a ratherlarge sum for a test, food producers are restricted by therelatively low food price. Hence, the hope is to come up with aminiaturized device that can accommodate both the sampleprep and the subsequent detection steps in a single device,indeed in a nano-volume chamber that obviates the cultivationcompletely. A truly viable alternative to the microbial enrichmentstep is the long needed revolution we all are waiting for. Andthere are some indications that this future is not that far away.

CONFIRMED:Jeffrey Hoorfar, Professor, Division of Food Microbiology,National Food institute, Technical University of Denmark(DTU), Denmark

Development and Use of Strategies for Detection ofMicrobial Pathogens (Including Virus and Bacteria) inFood and Environmental Samples • Review and state of the art in strategies for rapidmolecular methods in foodstuff

• Role of control for final assessment of results• Quantification: myth or reality?

CONFIRMED:David Rodríguez-Lázaro, Assistant Professor ofMicrobiology, University of Burgos, Spain

15.40 - 16.05 Circulating MicroRNAs Measured by Droplet Digital PCR asBiomarkers of Pancreatic Islet Cell Destruction• Biomarkers for monitoring pancreatic islets destructionin type 1 diabetes (T1D) are lacking

• Using droplet digital PCR we measured the amount ofmiRNAs of pancreatic origin in plasma at sequentialtime points after pancreatic islet transplantation inhumans

• Our result show that circulating miRNAs measured byddPCR are promising biomarkers of islet destruction

CONFIRMED:Vito Lampasona, Senior Scientist, Clinical MolecularBiology, Center for Translational Genomics andBioinformatics, San Raffaele Scientific Institute, Italy

qPCR for Quantitative Detection and Typification of DairyBacteriophagesWe have used the qPCR technology to quantifybacteriophages that infect two of the lactic acid bacteriaspecies mostly used in dairy industry: Lactobacillusdelbrueckii and Streptococcus thermophilus. We haveapplied the qPCR technology not only to quantify but alsoto identify in a single reaction, different bacteriophagesspecies present in a sample, such as the identification ofS. thermophilus bacteriophages as belonging to the cosor pac types.CONFIRMED:

Beatriz del Rio Lagar, Associate Researcher, Departmentof Technology and Biotechnology of Dairy Products,Group of Molecular Microbiology, Instituto de ProductosLacteos de Asturias (IPLA-CSIC), Villaviciosa, Spain



15.10 - 15.40 Afternoon RefreshmentsPoster Presentation Sessions



16.05 - 16.30 Single Tube Nested Real-Time PCR - The Knowns and the Unknowns

Nested PCR uses two pair of primers, outer and inner ones, in consecutive PCR amplification steps, which leads toincreased detection sensitivity of the target DNA. An adaptation is known in TaqMan real-time PCR format and,moreover, in single tube, with no need to open the vials, reducing the risk of contamination. While developing thisadaptation, the researchers formulated the rules about required primers' and probe melting temperatures, positionsand the PCR regimen. However, we have observed that these rules can be broken, while the PCR reaction staysfunctional and the effect of increasing sensitivity remains. This raises questions about the reaction mechanism. Ourexperience with single tube nested real-time PCR from developing the Cryptosporidium parvum (Minarovičová 2009)and peanut (Bergerová 2011) detection assays will be discussed.

CONFIRMED:Barbara Brezna, VÚP Food Research Institute, Bratislava, Slovakia

Chairman’s Closing Remarks and Conference Close



Plant Genomics Congress USA23 – 24 September, 2013

St Louis Missouri

Over 200 people from 23 countries attended the European Plant Genomics Congress earlier this year. This extraordinary

gathering provided delegates a unique opportunity to hear about the latest research in Plant Genomics from leaders at

the cutting edge of this field.

Building on the outstanding success of this event the Plant Genomics Congress USA is expected to attract 150 experts

working in next generation sequencing, plant genomics/ sciences, epigenetics, bioinformatics and data management,

the conference will examine the latest NGS platforms and technologies suitable for progressing plant based research as

well as tools to enable successful analysis.

For more details contact [email protected]

Microbiome / Microbiota R&D and Business Collaboration Forum7th – 8th October, 2013

San Diego, USA

Recent advances in understanding the influence of the Microbiome on human health have unequivocally demonstrated

the substantive impact of microbiome dysbiosis on the etiology of dozens of human diseases and syndromes including

Type-1 diabetes, refractory Clostridium difficile infections, ulcerative colitis, inflammatory bowel disease, Crohn’s,

necrotizing enterocolitis, celiac disease, asthma, pancreatic & colon cancers, childhood obesity, sinusitis and vaginosis,

among many others. As well, more disease associations are identified weekly in the literature.

Propitiously, the 5-year U.S. national Human Microbiome Project (HMP) was completed in June 2012, and all sequencing

data was placed in the public domain, thereby offering a rich source of free information for basic research scientists,

translational researchers, and biotech & pharma companies seeking new business opportunities.

This first-in-class, microbiome-focused hybrid R&D and business conference attracting 150 attendees from all over the

world, plus a 40-strong speaker faculty, will provide an interactive networking forum to both further research and

commercialization opportunities.

For more details contact [email protected]

New Events from Global Engage

Sponsorship offers a number of benefits including the opportunity to meet and form partnerships with delegates on aformal and informal basis in a relaxed and professional atmosphere. There are social networking opportunities duringrefreshment and lunch breaks held in our exhibition area and at cocktail receptions and gala dinners.

Global Engage sponsorship packages offer a wide range of attendance options to suit your budget and, as well asexhibiting and taking part in prearranged one-to-one meetings, you can also incorporate pre-event marketing, brandingand speaker presentations on the main conference agenda. For more information contact Steve Hambrook, ConferenceDirector, Global Engage Ltd. [email protected] +44 (0) 1865 849841

Conference Fees

Payment Details

Total: Cheques should be made out to: Global Engage Ltd. Please tick here to receive an invoice in advance of payment

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www.globalengage.co.uk/qpcr.html

VenueHilton Lyon70 Quai Charles De GaulleLyon, 69463France

AccommodationHotel accommodation is not included in your fee. To reserve aroom at the conference hotel, please send an email to Scott Taylorat [email protected].

Other detailsFull Terms & Conditions are set out at www.globalengage.co.uk

THE DELEGATE BOOKING FEE INCLUDES: all meals and refreshmentsthroughout the conference day, conference presentations, openworkshop and general panel sessions and networking/socialevents, conference and speaker notes. *Unless you have requestedthe one-to-one opt out fee you agree to participate in one-to-onenetworking meetings.

QUESTIONNAIRE: Each Delegate must complete and return a“Personal and Company Details Questionnaire” issued by theOrganiser on receipt of the Delegate Booking Form.

CONFIRMATION: If you have not received confirmation of yourbooking prior to the event, please call Global Engage on +44 (0)1865 849841. Your delegate place is not confirmed until paymentis received. Payment must be received before the conference date.If payment has not been received before the conference dateGlobal Engage reserves the right to ask for a credit or debit cardguarantee of payment when you register at the conference.

*BANK TRANSFER PAYMENTS: When paying by Bank Transfers quotethis reference: qPCR1

(Please ensure ALL bank charges are met by your organisation)

CANCELLATIONS/SUBSTITUTIONS: Delegates cancelling more than onecalendar month prior to event receive a full refund, one calendarmonth or less prior to event there is no refund. A substitutedelegate of equal standing can be nominated within a week ofthe event and must be approved by the Organiser in advance inorder to avoid cancellation charges.

ORDER CONFERENCE DOCUMENTATION:I cannot attend the conference but wish to buy the event docu-mentation pack, which includes the speakers’ presentations Full documentation costs £199 + VAT.To order, complete the registration form and method of payment.Payment must be received before the documentation andpassword can be despatched.

PROGRAMME CHANGES:Global Engage reserves the right to make any necessary alterations/changes to the programme.Personal Data is gathered in accordance with the Data ProtectionAct 1998.If you do not wish to receive promotional material from GlobalEngage, please tick here If you do not wish to receive promotional material from the EventSponsors, please tick here If you do not wish to receive promotional material from any other3rd party, please tick here Please return this form with the address and customer code, clearlyvisible if you wish us to remove your records from our database.

Industry Delegate €899

Academic Delegate €595

Solution Provider €995

Mail: Global Engage, Suite B, The Kidlington

Centre, Kidlington, Oxfordshire OX5 2DL,

United Kingdom.

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qPCR & Digital PCR Congress · 2015-04-24 · separate biological variation from technical one. In this talk, I will address some of the key issues in breadth of application, sensitivity