Isolation of DNA, RNA, and proteins

Sample & Assay Technologies

Simplify Complexity

Isolation of DNA, RNA, and proteins from archived t issues for molecular pathology studies

Sample & Assay Technologies- 2 -

Agenda

� Tissue Management at QIAGEN

� FFPE Product Portfolio

� 2 D SDS-PAGE from FFPE tissues

� Standardization with PAXgene Tissue


QIAGEN‘s Tissue Management Technologies

NEW: PAXgeneTissue System

Formalin Fixation

RNA/miRNA DNA Protein Morphology

= Two options for successful analyses !


Agenda






.Advantages of FFPE in Clinical Practice

� Vast Archive of FFPE Samples � 1Bio archived / + 20 Mio per year

� Established Clinical Procedure � Supports therapeutic decision

making� Stainings possible (IHC or FISH)

Formalin-Fixed Paraffin-Embeded (FFPE) Tissue


FFPE Samples in Molecular PathologyTechnical Challenges

.Formaldehyde fixation results in cross linking of:� Protein - protein

� Protein - nucleic acid

� Nucleic acid - nucleic acid

.Paraffin embedding results in:� Incubation at >60°C results in nucleic acid fragmentati on

� Deparaffination is required prior to nucleic acid preparation

.-> Quality of nucleic acids from FFPE samples is compromised due to� Formaldehyde crosslink / modification

� Fragmentation (heat + buffer conditions)

� interference of residual formaldehyde with enzymatic assays (RT, PCR)


The effects of crosslinksFixation of tissue using formaldehyde

No Fixation Formaldehyde Fixation Prolonged FA Fixation

Easy extraction and amplification of nucleic

acids and proteins

Challenging extraction and amplification of nucleic

acids and proteins

Limited extraction and amplification of nucleic

acids and proteins


� DNA � QIAamp DNA FFPE Kit� EZ1 DNA FFPE Kit (Automated)� Repli-g FFPE (Whole Genome Amplification)

� RNA � RNeasy FFPE Kit� miRNeasy FFPE Kit

� Protein � Qproteome FFPE Tissue Kit� Qproteome FFPE 2D-PAGE Kit

� Amplification� Quantifast SYBR Green PCR Kit� Quantifast Probe PCR Kit� miScript PCR System

Unlocking FFPE Tissue ArchivesThe largest portfolio of specific solutions for FFP E based research

Photo REMP

DNA RNA Protein

Genomics Gene Expression Protein Expression

Automatable on QIAcube!

Automatable on QIAcube!


Qproteome FFPE Tissue Kit Procedure

FFPE FFPE FFPE FFPE tissuetissuetissuetissue sectionssectionssectionssections

RemoveRemoveRemoveRemove paraffinparaffinparaffinparaffinand and and and rehydraterehydraterehydraterehydrate

AddAddAddAdd ExtractionExtractionExtractionExtraction bufferbufferbufferbuffer EXBEXBEXBEXBfollowedfollowedfollowedfollowed bybybyby heatheatheatheat treatmenttreatmenttreatmenttreatment

FFPE FFPE FFPE FFPE tissuetissuetissuetissue

Transfer Transfer Transfer Transfer supernatantsupernatantsupernatantsupernatantintointointointo a a a a newnewnewnew tubetubetubetube afterafterafteraftercentrifugationcentrifugationcentrifugationcentrifugation

ExtractedExtractedExtractedExtracted ProteinProteinProteinProtein

J Pathol. 2007 Feb;211(3):370-8; Becker KF, et al


Quantitative extraction of intact proteins Results comparable to protein extracted from Fresh and frozen tissue

66.3-55.4-

36.5-31-

200-

14.4-

116.3-

21.5-F

roze

n

FF

PE

Con

trol

� ß-Actin

66.3-55.4-

36.5-31-

200-

14.4-

116.3-

21.5-

Fro

zen

FF

PE

Con

trol

� ß-Actin

Fro

zen

FF

PE

Con

trol

Brain (Frozen vs FFPE)Colon (Frozen vs FFPE) Brain (Frozen vs FFPE)

Comparable yields can be achieved from FFPE material vs fresh frozen tissueProteins are of full length and post-translational modifications are preserved


.HER2 is a proto-oncogene that shows

overexpression in different cancer types.

.Aberration rates correlate with individual patient

prognosis and are important predictive markers for targeted therapy.

.A: HER2 was extracted with the Qproteome FFPE

Tissue kit and detected by Western blotting and

protein microarrays.

.B: HER2 gene amplification was confirmed by fluorescence in situ hybridization (FISH).

A

B

Increased HER2 expression and thus immunoreactivity

Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis. Becker KF, Schott C, Hipp S, Metzger V, Porschewski P, Beck R, Nahrig J, Becker I, Hofler H. J Pathol. 2007 Feb;211(3):370-8

HER2 overexpression and gene amplificationas markers in breast cancer diagnostics


Agenda






Proteomics Based Clinical PathologyIdeal Workflow for Proteomics Analysis of FFPE Tiss ue

Routine Histology(FFPE)

ProteinExtraction

Protein array orSDS-PAGE

Antibody baseddetection

Quantification

Clean upfor 2D

Spotpicking

MS-analysis


Literature: “It is not possible to analyze FFPE Sample o n 2D PAGE“

� So far no succesful report of 2D-PAGE in the literature

� Ahram M. et al. : „ … The presence of artifacts in the form of vertical smears was

also more prominent in 2-D gels of ethanol-fixed tissues than of frozen tissues. This

artifacts could be caused by the presence of nucleic acids or remanant paraffin in the

samples. …“ (no result was obtained with formalin-fixed, paraffin-embedded tissue)

� Hood B.L. et al.

FFPE frozen EtOH fixed


Qproteome FFPE 2D PAGE Tissue Kit Procedure


Reproducible extraction of proteins from FFPE samples prior to 2D PAGE

� Extraction of proteins from FFPE liver (rat); 4 samples, serial sections

� 2D PAGE – Analysis (sample 1 and 2)

Actin Erk2

1 2

A_226 Sample & Assay Technologies- 17 -

Spot # IPI entryMascot score

ProteinMW [kDa]

pI

1 IPI00191737 477 Alb Serum albumin precursor 70,6 6,092 IPI00551812 406 Atp5b ATP synthase subunit beta, mitochondrial precursor 56,3 5,193 IPI00765011 474 LOC295810 similar to Actin, cytoplasmic 2 59,1 5,674 IPI00389611 196 Rgn Regucalcin 34,0 5,275 IPI00231742 417 Cat Catalase 60,0 7,076 IPI00396910 136 Atp5a1 ATP synthase subunit alpha, mitochondrial precursor 59,8 9,227 IPI00214480 217 Fah Fumarylacetoacetase 46,2 6,678 IPI00201413 499 Acaa2 3-ketoacyl-CoA thiolase, mitochondrial 42,2 8,099 IPI00205332 460 Etfa Electron transfer flavoprotein subunit alpha, mitochondrial precursor 35,2 8,6210 IPI00760117 273 Comt Isoform 2 of Catechol O-methyltransferase 25,0 5,1111 IPI00231013 439 Cyb5 Isoform Short of Cytochrome b5 11,4 5,2612 IPI00231643 587 Sod1 Superoxide dismutase 16,0 5,8813 IPI00231292 241 Hrsp12 Ribonuclease UK114 14,5 6,2114 IPI00230788 477 Ca3 Carbonic anhydrase 3 29,7 6,8915 IPI00411230 298 Gstm2 Glutathione S-transferase Mu 2 25,8 6,9016 IPI00411230 540 Gstm2 Glutathione S-transferase Mu 2 25,8 6,9017 IPI00231639 494 Gstm1 Glutathione S-transferase Mu 1 26,0 8,2718 IPI00325189 372 Nme2 Nucleoside diphosphate kinase B 17,3 6,9219 IPI00190790 433 Fabp1 Fatty acid-binding protein, liver 14,3 7,7920 IPI00679202 150 Tf Isoform 1 of Serotransferrin precursor 78,5 7,14

Protein Identification by MS

-> Proteins of different sizes, pIs and cellular localization identified


List of publications featuring the Qproteome FFPE Ki t

� Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis. Becker KF, Schott C, Hipp S, Metzger V, Porschewski P, Beck R, Nahrig J, Becker I, Hofler H. J Pathol. 2007 Feb;211(3):370-8

� Guided protein extraction from formalin-fixed tissues for quantitative multiplex analysisavoids detrimental effects of histological stains. Karl-Friedrich Becker, Christina Schott, Ingrid Becker and Heinz Höfler. Proteomics Clin. Appl. 2008, 2, 737–743

� Extraction of Phosphorylated Proteins from Formalin-Fixed Cancer Cells and Tissues. Karl-Friedrich Becker, Hildegard Mack, Christina Schott, Susanne Hipp, Anne Rappl, Guido Piontek and Heinz Höfler. The Open Pathology Journal, 2008, 2, 46-52

� Precise measurement of the E-cadherin repressor Snail in formalin-fixed endometrialcarcinoma using protein lysate microarrays. Susanne Hipp, Axel Walch, TiborSchuster, Heinz Höfler, Karl-Friedrich Becker. Clin Exp Metastasis. 2008;25(6):679-83. Epub 2008 Feb 29.

� Isolation of high quality protein samples from punches of formalin fixed and paraffinembedded tissue blocks. J. Kroll, K.F. Becker, S. Kuphal, R. Hein, F. Hofstädter and A.K. Bosserhoff. Histol Histopathol (2008) 23: 391-395


Agenda






QIAGEN‘s Tissue Management Technologies

NEW: PAXgeneTissue System

Formalin Fixation

RNA/miRNA DNA Protein Morphology

= Two options for successful analyses !


Tissue Repositories – Standardization is Key

. Challenges:� Sample to sample variation – we need more

standardization of � Sample collection� Archiving methods� Processing� Quality standards

� Controls� Cost

� Significant upfront and maintenance� Biobank-derived information triggers

expensive projects� Limited comparability of assay results

� Inter-site� Intra-site� Content targets/analytical methods change

.

Standardization is key!


The effects of crosslinksFixation of tissue using formaldehyde

No Fixation Formaldehyde Fixation Prolonged FA Fixation

Easy extraction and amplification of nucleic

acids and proteins

Challenging extraction and amplification of nucleic

acids and proteins

Limited extraction and amplification of nucleic

acids and proteins


Overcoming FFPE Limitation in prospective studiesPAXgene Tissue Workflow

DNA

RNA

miRNA

IHC

H&EPAS

WesternBlot

ISH

ProcessStabilizeFixPlace in

histo-cassette

PFPE*

*PAXgene Tissue fixed paraffin embedded*PAXgene Tissue fixed paraffin embedded

Resect

Allows histomorphology and molecular testingfrom the same specimen


Overcoming FFPE LimitationPAXgene Tissue System Components

PAXgene Tissue Container

� Tissue Fixation� Tissue Stabilization� Process Standardization

� RNA/miRNA/DNA purification from slides (coming soon)� RNA/miRNA/DNA purification after staining and microdissection (coming soon)� Protein Extraction: Using Qproteome FFPE Kit

� RNA� miRNA Purification� DNA

PAXgene Tissue Kits

PAXgene Tissue Supplementary Protocols


PAXgene Tissue FeaturesPreservation of Morphology and DNA

PFPE(PAXgene Tissue)

FFPE(NBF*)

Live

r

PFPE FFPE

20

21

22

23

24

25

26

CT

DNA

qPCRßactin TaqManPrimer/Probe

8-plex PCR, single copy genes222 to 955bp

Gel electrophoresis

* Neutral Buffered Formalin


PAXgene Tissue Protein ExtractionFrozen versus PFPE

Sample 1 2 3 4 5 6

� Protein extractin from rat liver, intestine, brain� 10 mg tissue snap frozen in liquid nitrogen� 3x 10 µm thick sections of PFPE (PAXgene Tissue fixed paraffin-embedded) tissue

� SDS-PAGE with 10 µg protein per lane� Western blotting with anti-Actin Antibody� Ponceau staining

250130

9572

55

36

28

17

Sample 1 2 3 4 5 6

- actin (48 kDa)

BrainPFPE6

IntestinePFPE5

LiverPFPE4

BrainSnap frozen3

IntestineSnap frozen2

LiverSnap frozen1

Tissuetype

FixationID

Western Blot Ponceau Staining

PFPEFrozen PFPEFrozen


Thank you

Documents

Isolation of DNA, RNA, and proteins