QIAGEN Driving Innovation in Epigenetics

Transcription

1 QIAGEN Driving Innovation in Epigenetics EpiTect and PyroMark A novel Relation setting Standards for the reliable Detection and accurate Quantification of DNA-Methylation May 2009 Gerald Schock, Ph.D. Senior Global Product Manager Epigenetics & WGA - 1 -

2 QIAGEN Driving Innovation in Epigenetics Introduction Epigenetics mechanisms Analyzing DNA methylation Major steps in DNA methylation analysis Bisulfite Conversion of gdna Whole Bisulfitome Amplification High Resolution Melting Analysis Principle of HRM Assay Design Increasing accuracy of HRM assays Pyrosequencing Analyzing consequtive CpG sites Principle of Pyrosequencing Workflow Built-in Quality Control Controls in Methylation Analysis - 2 -

3 Epigenetics Definition and Mechanisms.Epigenetics..The study of reversible inheritable influence on gene activity that is not accompanied by a change in the DNA sequence.epigenetics mechanisms DNA Methylation Histone Modification micro RNA Figures taken from National Institute of General Medical Sciences

4 Epigenetics DNA Methylation.DNA methylation Adding of a methyl group on cytosine residues of CpG dinucleotides Reversible process.cpg islands High frequency of CpG dinucleotides: One CpG per 10 nucleotides CpG islands are often found in promoter regions CpG islands co-localize with 60% of all promoters.function Genes in methylated DNA are silenced In humans 70-80% of CpGs are methylated Most non methylated CpGs are found in regulatory elements Relevance on Cancer Hypermethylation and silencing of important tumour suppressor genes Hypomethylation and activating of oncogenes (e.g. K-ras, C-myc) - 4 -

5 DNA Methylation Analysis Pre-Analytical and Analytical Steps Sample collection and stabilization DNA purification Bisulfite treatment Analysis Preanalytical Epigenetics Analytical Epigenetics Blood Biopsies Cells Paraffin slides gdna Bisulfite treatment converts unmethylated C into U Converted DNA Cloning & sequencing Methylation specific Primer (MSP) PCR Bisulfite sequencing Pyro- Sequencing gel-based MSP real-time MSP HRM - 5 -

6 DNA Methylation Analysis Bisulfite Conversion Sample collection and stabilization DNA purification Bisulfite treatment Analysis Why is bisulfite treatment required?.bisulfite converts all unmethylated cytosines into uracil Methylated cytosines in CpG sequences remain unaffected Uracil is replaced by thymine during PCR reaction.bisulfite converted DNA can then be analyzed - 6 -

7 DNA Methylation Analysis Bisulfite Conversion Sample collection and stabilization DNA purification Bisulfite treatment Analysis Why does conversion efficiency matter?.risk of incomplete bisulfite conversion Unmethylated cytosines remain as cytosines False positive methylation results Incorrect data interpretation - 7 -

8 DNA Methylation Analysis Bisulfite Conversion Sample collection and stabilization DNA purification Bisulfite treatment Analysis Why does conversion efficiency matter?.risk of incomplete bisulfite conversion Unmethylated cytosines remain as cytosines False positive methylation results Incorrect data interpretation Unmethylated DNA PCR specific for methylated DNA Complete Complete bisulfite bisulfiteconversion increases increases the the reliability reliability of of methylation methylation analysis analysis - 8 -

9 DNA Methylation Analysis Bisulfite Conversion Sample collection and stabilization DNA purification Bisulfite treatment Analysis EpiTect Bisulfite Technology Unique DNA Protect Mechanism Facilitates formation of single stranded DNA Ensures complete cytosine conversion Minimizes DNA fragmentation during bisulfite conversion reaction conversion rate determined by bisulfite sequencing DNA Protect Buffer 99.4% 99.8% Confirmed by Solexa and 454 Sequencing* Eliminates false positive methylation results - 9 -

10 Meissner et al 2008 Nature Conversion efficiency determined by Solexa Genome Analyzer

11 Taylor et al 2008 Cancer Res 67 Conversion efficiency determined by 454 Sequencing

12 EpiTect Bisulfite Technology Sensitive Reaction from Small Sample Amounts.Highly sensitive reaction As little as 1 ng template DNA Wide DNA Input range: 1 ng 2 µg DNA from e.g. Blood, Serum, FFPE-tissue, Biopsies Protection of DNA degradation: Detection of even long PCR fragments (Grunau et al. NAR; 29, 65: Limit PCR amplicon bp) Marker Control 1 µg 100 ng 10 ng 1 ng Control 1 µg 100 ng 10 ng 1 ng Marker Experimental setup: Human genomic DNA was purified from blood (QIAamp DNA Blood Mini Kit) 150 bp bp Various amounts (1 ng 1 µg) were converted using the EpiTect Bisulfite Kit PCR was performed using 5% of the eluate and the HotStarTaq Master Mix 2 sets of primers were used, designed to amplify converted DNA only

13 EpiTect Bisulfite Technology DNA Long Term Storage.Efficient removal of bisulfite salt long-term storage of converted DNA Allows storage of the converted DNA for at least 12 months* Experimental setup: DNA converted using the EpiTect Bisulfite Kit and stored at 20ºC for 12 months Compared to freshly converted DNA in a real-time PCR assay using the QuantiTect Probe Kit and converted DNA specific primers *Ongoing experiment. Actual data are available on request

14 DNA Methylation Analysis Bisulfite Conversion - Summary Sample collection and stabilization DNA purification Bisulfite treatment Analysis PAXgene Blood DNA Kits Allprotect Tissue Reagent QIAamp Kits DNeasy Kits EpiTect Bisulfite Kits EpiTect Bisulfite Kits DNA Protect Technology Ensures complete bisulfite conversion Minimizes DNA fragmentation Reliability & Sensitivity Optimized protocols for precious samples FFPE samples highly diluted DNA EpiTect EpiTect Bisulfite Bisulfite Kits Kitscombine complete complete conversion conversion of of methylated methylated DNA DNA in in less less than than 6 hours hours with with an an innovative innovative protection protection against against DNA DNA degradation degradation for for fast fast and and reliable reliable results. results

15 DNA Methylation Analysis Low amounts of DNA - Bottlenecks & Challenges Sample collection and stabilization DNA purification Bisulfite treatment Amplification Analysis PAXgene Blood DNA Kits Allprotect Tissue Reagent QIAamp Kits DNeasy Kits EpiTect Bisulfite Kits EpiTect Whole Bisulfitome Kit Limited Methylation Analysis Low DNA Low bisulfite converted DNA Insufficient for all analysis Amplification (WGA) of bisdna is critical altered nucleotide composition due to bisulfite conversion smaller DNA fragment size

16 EpiTect Whole Bisulfitome Amplification Proven amplification procedure and easy one step reaction setup EpiTect Whole bisulfitome amplification (WBA) procedure Based on Multiple Displacement Amplification (REPLI-g technology) Isothermal amplification / Strand displacement High processivity of Phi29 polymerase for unbiased DNA replication Optimized for bisulfite converted DNA Yields 1-3 µg bisulfite converted DNA (50-fold increase in the DNA amount) Principle of Multiple Displacement Amplification (MDA)

17 EpiTect Whole Bisulfitome Amplification Reliability in Real-Time PCR Real-time PCR analysis of four different loci Starting amount: 10 ng bisulfite converted DNA (-WBA) and whole bisulfitome amplified DNA (+WBA) Missing DNA protection in bisulfite conversion results in Fragmented DNA (high C T values) Comparable C T values after WBA with EpiTect Bisulfite converted DNA Less efficient WBA with nonprotected DNA (even higher C T values)! "!

18 EpiTect Whole Bisulfitome Amplification Reliability in End-Point PCR Reliable end-point PCR Starting amount: 50 ng genomic DNA bisulfite converted in 6 independent reactions using the EpiTect Bisulfite Kit WBA: 10 ng bisulfite converted DNA using EpiTect Whole Bisulfitome Kit PCR bands obtained are comparable in size and intensity to original bis-dna

19 DNA Methylation Analysis Whole Bisulfitome Amplification - Summary Sample collection and stabilization DNA purification Bisulfite treatment Amplification Analysis PAXgene Blood DNA Kits Allprotect Tissue Reagent QIAamp Kits DNeasy Kits EpiTect Bisulfite Kits EpiTect Whole Bisulfitome Kit EpiTect Whole Bisulfitome Kit Increases DNA amount after bisulfite conversion Dedicated WBA-technology using proven REPLI-g technology More bisdna for more methylation Analysis Reliable amplification Proven REPLI-g technology using gold standard MDA technology EpiTect EpiTect Whole Whole Bisulfitome BisulfitomeKit Kit overcomes overcomes limitations limitations in in methylation methylation analysis analysis derived derived from from low low amounts amounts of of DNA. DNA. It It is is the the first first dedicated dedicated kit kit for for the the amplification amplification of of the the entire entire bisulfite bisulfite converted converted genome genome (Bisulfitome). (Bisulfitome). * Not suitable for DNA derived from FFPE samples

20 DNA Methylation Analysis Analysis Sample collection and stabilization DNA purification Bisulfite treatment Analysis PAXgene Blood DNA Kits Allprotect Tissue Reagent QIAamp Kits DNeasy Kits EpiTect Bisulfite Kits EpiTect Whole Bisulfitome Kit Detection of converted DNA: Methylation-specific PCR (MSP) gel-based / Real-Time Pyrosequencing Bisulfite sequencing High Resolution Melting (HRM)

21 Epigenetics Methodologies Overview Positioning of various available techniques Number of targets HRM High Resolution Melt Pyrosequencing Methylation specific PCR (MSP) MethyLight Sequence Information provided Screening/Detection CpG Analysis Specific Quantification Screening for unknown CpG positions Analyze specific CpG positions Analyze & Quantify specific CpG sites

22 HRM & Pyrosequencing Used at different stages within a project Sample collection & Stabilization DNA purification Bisulfite Treatment /Amplification Analysis.HRM: Screening of hundreds of cells or targets to identify regions (e.g. CpG islands) of interest methylated CpG site unmethylated CpG site inactive Gene.Pyrosequencing: CpG specific and quantitative analysis of those regions to understand gene regulation by DNA methylation Promotor region Gene active Gene Legend Reference Sample CpG island A CpG island A CpG island B CpG island C CpG island Z CpG island B CpG island C CpG island Z Identification (HRM) CpG island C Identify the CpG island of interest Detailed analysis (Pyro sequencing) 66% 33% Sample 1 Sample 2 Sample 3 Detailed CpG-specific analysis with more samples (in tissue or cells)

23 HRM & Pyrosequencing Used at different stages within a project Sample collection & Stabilization DNA purification Bisulfite Treatment /Amplification Analysis.HRM: Screening of hundreds of cells or targets to identify regions (e.g. CpG islands) of interest methylated CpG site unmethylated CpG site inactive Gene.Pyrosequencing: CpG specific and quantitative analysis of those regions to understand gene regulation by DNA methylation Promotor region Gene active Gene Legend Reference Sample CpG island A CpG island A CpG island B CpG island C CpG island Z CpG island B CpG island C CpG island Z Identification (HRM) CpG island C Identify the CpG island of interest Detailed analysis (Pyro sequencing) 66% 33% Sample 1 Sample 2 Sample 3 Detailed CpG-specific analysis with more samples (in tissue or cells)

24 Principle of HRM Assay Design Example: Promoter of APC Bisulfite conversion & PCR Melting analysis Fluorescence T A C G Increasing temperature PCR primer Region of interest C m C m C CpG island PCR primer C Bisulfite conversion U PCR T

25 EpiTect HRM PCR Kit Reliable HRM results without optimization Quantification of methylation degrees by HRM Samples are compared to HRM results obtained with mixtures of unmethylated DNA and methylated DNA (EpiTect Control DNA) A CpG island from the promoterregion of the APC gene (adenomatosis polyposis coli) was amplified. Standard [Mg 2+ ] supplied in the mastermix was used

26 EpiTect HRM PCR Kit Reliable HRM results without optimization Quantification of methylation degrees by HRM Run sample and compare to HRM results obtained with mixtures of unmethylated DNA and methylated DNA (EpiTect Control DNA) A CpG island from the promoterregion of the APC gene (adenomatosis polyposis coli) was amplified. Standard [Mg 2+ ] supplied in the mastermix was used

27 Rotor-Gene Q Features ensuring accurate HRM results Rotor-Gene Q. High-intensity & high sensitivity optics Detection of small changes in fluorescence. Very precise temperature control Allow small increments and thereby higher resolution. Near-perfect temperature uniformity Well-to-well uniformity +/ C Centrifugal fan drives air around chamber Chamber vent opens expelling hot air Centrifugal fan Drives air into chamber Cool air in

28 Rotor-Gene Q Features ensuring accurate HRM results Rotor-Gene Q. High-intensity & high sensitivity optics Detection of small changes in fluorescence. Very precise temperature control Allow small increments and thereby higher resolution. Near-perfect temperature uniformity Well-to-well uniformity +/ C. High-speed data capture Small increments = lots of data points Tubes Spin in Rotor (Red) Reaction Chamber Spindle/Motor Assembly Le ns Detection Filters PMT Detector Assembly LED Light Source Assembly

29 EpiTect HRM PCR Kit & Rotor-Gene Q Proof data M U U U M M Roche HRM Kit LC 480 QIAGEN HRM Kit LC 480 QIAGEN HRM Kit Rotor-Gene Q Non specific amplification Problems to distinguish small changes of methylation differences EpiTect HRM PCR Kit with Rotor-Gene Q generates superior results

30 HRM & Pyrosequencing Used at different stages within a project Sample collection & Stabilization DNA purification Bisulfite Treatment /Amplification Analysis.HRM: Screening of hundreds of cells or targets to identify regions (e.g. CpG islands) of interest methylated CpG site unmethylated CpG site inactive Gene.Pyrosequencing: CpG specific and quantitative analysis of those regions to understand gene regulation by DNA methylation Promotor region Gene active Gene Legend Reference Sample CpG island A CpG island A CpG island B CpG island C CpG island Z CpG island B CpG island C CpG island Z Identification (HRM) CpG island C Identify the CpG island of interest Detailed analysis (Pyro sequencing) 66% 33% Sample 1 Sample 2 Sample 3 Detailed CpG-specific analysis with more samples (in tissue or cells)

31 DNA Methylation Analysis Why analyzing CpG methylation in detail? Methylation patterns in genomic DNA change due to factors such as environment, age, disease (cancer and others), etc. These changes can affect All CpG sites (e.g. change from unmethylated CpGc to methylated CpGs) Normal Gene expression Promoter (CpG Island) Intergenic Region Exon of Regulatory Gene Cancer

32 DNA Methylation Analysis Why analyzing CpG methylation in detail? Methylation patterns in genomic DNA change due to factors such as environment, age, disease (cancer and others), etc. These changes can affect All CpG sites (e.g. change from unmethylated CpGc to methylated CpGs) Only some CpG sites (resulting in a mosaic of methylated and unmethylated CpGs) Normal Gene expression Promoter (CpG Island) Intergenic Region Exon of Regulatory Gene Cancer I Cancer II

33 DNA Methylation Analysis Why analyzing CpG methylation in detail? Methylation patterns in genomic DNA change due to factors such as environment, age, disease (cancer and others), etc. These changes can affect All CpG sites (e.g. change from unmethylated CpGc to methylated CpGs) Only some CpG sites (resulting in a mosaic of methylated and unmethylated CpGs) Methylation pattern in the tumorsupressor RASSF1A in neighboring CpG sites in 4 tumor samples (duplicate runs)

34 DNA Methylation Analysis Sequencing of bisulfite converted DNA Bottlenecks & Challenges Sample collection and stabilization DNA purification Bisulfite treatment Amplification Analysis PAXgene Blood DNA Kits Allprotect Tissue Reagent QIAamp Kits DNeasy Kits EpiTect Bisulfite Kits EpiTect Whole Bisulfitome Kit PyroMark Q24 / Q96 PyroMark PCR Kit PyroMark Gold reagents EpiTect Control DNA Analyzing consecutive CpG sites Challenge: HRM only shows differences in the methylation status MSP requires exact sequence information of CpG islands to be analyzed Quantitative MSP and MethyLight requires several CpG sites for primer or probe Methylation of single CpG sites cannot be determined Sequencing data are needed Direct Sanger Sequencing does not give quantification results Cloned Sanger Sequencing is highly labor and time intensive

35 The Principle of Pyrosequencing Technology Assay Design.Assay design PyroMark Assay Design Software 2.0 PyroMark Assay Database Free Online Access for customers PyroMark RUO Kits If you can run a PCR, you can sequence with Pyrosequencing Jon Jonasson, University Hospital, Linköping, Sweden PCR primer Sequencing primer Region of interest PCR primer

36 The Principle of Pyrosequencing Technology Assay Design using PyroMark Assay Design Software 2.0.Assay design PyroMark Assay Design Software 2.0 Step 1 Step 2 Step 3 Dedicated functionality for CpG assay design Step 1: Enter original sequence Software converts sequence into bisulfite treated sequence Software identifies all CpG sites and marks these in bold Software presents possible bisulfite conversion controls in color Step 2: Select target region with sites of interest. Press run Step 3: Select assay design based on quality scoring

37 The Principle of Pyrosequencing Technology Assay Design using PyroMark Assay Design Software

38 The Principle of Pyrosequencing Technology Assay Design using PyroMark Assay Design Software

39 The Principle of Pyrosequencing Technology Assay Design using PyroMark Assay Design Software

40 The Principle of Pyrosequencing Technology Assay Design using PyroMark Assay Design Software

41 The Principle of Pyrosequencing Technology PCR.Assay design.pcr Amplify relevant region by PCR ( bp) One primer is biotinylated PCR primer Sequencing primer Region of interest PCR primer

42 The Principle of Pyrosequencing Technology PCR using PyroMark PCR Kit.Assay design.pcr PyroMark PCR Kit Larger signal heights More reliable quantification results (e.g. blue vs. red ) Temperature 10 µl PyroMark PCR Kit Temperature 10 µl Standard Hotstart PCR

43 The Principle of Pyrosequencing Technology Sample preparation.assay design.pcr.sample preparation Immobilize biotinylated PCR products onto streptavidin coated beads Separate strands by denaturation in NaOH Wash /neutralize the immobilized strand Anneal sequencing primer

44 The Principle of Pyrosequencing Technology Sample preparation.assay design.pcr.sample preparation.pyrosequencing One nucleotide (dntp) is added at a time Nucleotide incorporation generates Pyroposhpate (PP i ) Pyroposhpate (PP i ) is converted into light seen as peak in the Pyrogram trace Excess nucleotide is degraded before addition of the next base. Light Time

45 The Principle of Pyrosequencing Technology Analyzing a pyrogram for DNA-methylation.Sequence to be analyzed:.after Bisulfite conversion:.a G T T A C G A C.A G T T A C m G A C.and.A G T T A C G A C.A G T T A C G A T.and.A G T T A T G A T.Analyzed sequence: CpG methylation level:.a.g.t.t.a.c.g.a.t.27%.nucleotides added:.a.g.t.a.t.c.g.a.c.t.built-in Quality control: Successful Bisulfite conversion

46 The Principle of Pyrosequencing Technology Generating quantitative data in a sequence context.assay design.pcr.sample preparation.pyrosequencing.data analysis Sequence and quantitative information Allows complex analyses including DNA Methylation and heterozygote analysis PyroMark Q-CpG Software dedicated software for CpG methylation analysis Pyrogram provides quantitative CpG methylation in a sequence context

47 Pyrosequencing Accuracy - Linear response in measured methylation Linear response of methylation measured by Pyrosequencing in PCR-amplified products generated from controlled dilutions of in-vitro methylated (IVM) genomic DNA with unmethylated DNA (IVM DNA is 80% methylated)

48 Pyrosequencing Rapid results compared to classical sequencing methods Reproducible quantification of all analyzed sites PCR PSQ* *PSQ: Pyrosequencing Only semiquantitative PCR Direct Sanger Sequencing Highly labour intensive PCR Cloning Cloned Sanger Sequencing 2 h 3h 24h >3-5 days Time Time saving saving Fast Fast and and easy easyresults

49 Pyrosequencing Data Output and Built-in Quality Control of Bisulfite Conversion RASSF1A gene Before bisulfite treatment Analyzed sequence Built-in Quality Control Any C not followed by a G severs as quality control for the bisulfite reaction

50 Pyrosequencing PyroMark Q24 - Workflow Fast and easy workflow 24 samples analyzed in parallel in as little as 15 minutes after PCR PCR PCR Sample Prep Pyrosequencing ~ 2h ~ 10 min ~ 15 min

51 Pyrosequencing CpG methylation studies with PyroMark TM Q24 CpG Methylation Studies Any single CpG site Multiple consecutive CpG sites One gene at a time Several genes in the same analysis (analyze up to 24 different assays in one run) Global methylation level Estimate the global methylation levels using repetitive elements (LINE-1)

52 PyroMark product line Product Offering Overview Software PCR Kits, Reagents, Assay Kits Instruments PyroMark Q96 MD PyroMark Q24 Workstation Sample preparation PyroMark Q24 PyroMark Q96 ID

53 DNA Methylation Analysis Sequencing of bisulfite converted DNA Sample collection and stabilization DNA purification Bisulfite treatment Amplification Analysis PAXgene Blood DNA Kits Allprotect Tissue Reagent QIAamp Kits DNeasy Kits EpiTect Bisulfite Kits EpiTect Whole Bisulfitome Kit PyroMark Q24 / Q96 PyroMark PCR Kit PyroMark Gold reagents EpiTect Control DNA Pyrosequencing Highly sensitive methylation quantification combined with sequencing Real Sequence data - Methylation levels are presented in sequence context Variation in the methylation of various sites can be detected with high accuracy Methylation analysis can be combined with SNP typing in one assay Fast procedure and flexible throughput 1-96 samples analyzed in parallel in 1 hour after PCR Pyrosequencing Pyrosequencing The The Gold Gold standard standard for for accurate accurate and and sensitive sensitive methylation methylation analysis analysis that that combines combines quantification quantification analysis analysis with with highly highly reliable reliable sequencing sequencingdata. data

54 DNA Methylation Analysis Controls in Methylation Analysis Bottlenecks & Challenges Sample collection and stabilization DNA purification Bisulfite treatment Amplification Analysis PAXgene Blood DNA Kits Allprotect Tissue Reagent QIAamp Kits DNeasy Kits EpiTect Bisulfite Kits EpiTect Whole Bisulfitome Kit HRM Pyrosequencing EpiTect Control DNA Control Experiments Time constraints Availability of fully unmethylated genomic DNA (U-DNA) Preparation of fully methylated DNA (M-DNA) Standardization & Reliability Ensure complete bisulfite conversion of U- and M-DNA

55 Control PCR Experiments in Methylation Analysis Validation of methylation assays A Validation of M-primers B Validation of U-primers Forward M-primer %$ #$ Forward U-primer %$ #$ C %$ Validation of M-primers on genomic DNA Forward M-primer #$ %$ #$ #$ %$ Reverse M-primer %$ #$ #$ %$ Reverse U-primer #$ %$ Reverse M-primer #$ %$ #$ %$ Requirements fully methylated DNA (M-DNA) / fully unmethylated DNA (U-DNA) / Genomic DNA Risk False validation results are obtained in case of Incomplete methylated DNA/unmethylated DNA Incomplete conversion of methylated DNA/unmethylated DNA

56 Control PCR Experiments in Methylation Analysis Validation of methylation assays A Validation of M-primers B Validation of U-primers Forward M-primer %$ #$ Forward U-primer %$ #$ C %$ Validation of M-primers on genomic DNA Forward M-primer #$ %$ #$ #$ %$ Reverse M-primer %$ #$ #$ %$ Reverse U-primer #$ %$ Reverse M-primer #$ %$ #$ %$ EpiTect Control DNA Control DNAs for validation of primer specificity used for methylation analysis Pre-bisulfite converted, completely unmethylated human genomic DNA Pre-bisulfite converted, completely methylated human genomic DNA Human genomic DNA (completely unmethylated)

57 EpiTect PCR Control DNA MSP Gel Analysis of p16 locus M M Reliable positive and negative controls for p16 MSP EpiTect Control DNA (Lane 1 + 4) 10 ng each Frozen lung tumor DNA samles About 10 ng each MSP results were confirmed by Pyrosequencing Data kindly provided by Triantafillos Liloglou, Roy Castle International Centre for Lung Cancer Research, Molecular Oncology Unit, Liverpool, UK. M: Marker 1: EpiTect Methylated Control DNA 2,3: known tumor DNA positives 4: EpiTect unmethylated Control DNA 5,6: known tumor DNA negatives back

58 EpiTect PCR Control DNA MALDI TOF analysis of the MP6 locus EpiTYPER MALDI-TOF analysis EpiTect Methylated Control DNA (QIA METH DNA) EpiTect Unmethylated Control DNA (QIA UNMETH DNA) Mixture of both (QIA HTX DNA) EpiTect Control DNA QIA METH DNA QIA UNMETH DNA QIA HTX DNA NTC.Data kindly provided by Hany Ezzeldin, Mayo Clinic, Rochester, USA. back

59 EpiTect PCR Control DNA Pyrosequencing analysis of the MGMT locus EpiTect Methylated Control DNA / Assay: MGMT-new_p1 97% 100% 97% 99% 94% 92% 97% 95% 84% 84% 100% 98% 100% 96% 95% 95% EpiTect Unmethylated Control DNA / Assay: MGMT-new_p1 1% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 4% 0% 0% 0%.Data kindly provided by Uwe Gerstenmaier, varionostic GmbH, Ulm, Germany. back

60 EpiTect PCR Control DNA Standards for quantitative real-time PCR Quantification standards for Methylation Assays Mixtures of EpiTect Methylated Control DNA and EpiTect Unmethylated Control DNA to obtain 100%, 90%, 50%, 10%, and 0% methylated DNA MethyLight assays for the human PITX2 gene Distinct C T values obtained for each methylation Control

61 DNA Methylation Analysis Controls in Methylation Analysis Sample collection and stabilization DNA purification Bisulfite treatment Amplification Analysis PAXgene Blood DNA Kits Allprotect Tissue Reagent QIAamp Kits DNeasy Kits EpiTect Bisulfite Kits EpiTect Whole Bisulfitome Kit HRM Pyrosequencing EpiTect Control DNA EpiTect Control DNA Simplifies validation of primer specificity used for methylation analysis Ready-to-use, pre-bisulfite converted U- and M-DNA Saves time and increases standardization Suitable for a variety of methylation analysis methods Endpoint MSP, Real-Time PCR, Pyrosequencing, HRM, MALDI-TOF analysis, and others EpiTect EpiTect PCR PCR Control Control DNAs DNAs Ready Ready to to use use pre-converted pre-converted human human control control DNAs DNAsfor for standardized standardized and and reliable reliable control control reactions reactions in in PCR PCR methylation methylation experiments experiments

62 DNA Methylation Analysis QIAGEN Solutions for the Complete Workflow Sample collection and stabilization DNA purification Bisulfite treatment Amplification Analysis PAXgene Blood DNA Kits Allprotect Tissue Reagent QIAamp Kits DNeasy Kits QIAcube/EZ1 QIAsymphony EpiTect Bisulfite Kits QIAcube EpiTect Whole Bisulfitome Kit EpiTect MSP Kit EpiTect MethyLight PCR Kit EpiTect MethyLight Assays Home made Assays* Rotor-Gene Q EpiTect HRM PCR Kit EpiTect Control DNA PyroMark Q24 / Q96 ID / MD PyroMark PCR/PyroMark Gold Standardized preanalytical solutions Sample collection DNA purification Bisulfite conversion Whole Bisulfitome Amplification Standardized analytical solutions Methylation specific PCR (MSP) Quantitative methylation analysis High Resolution Melting (HRM) Analysis Pyrosequencing Control DNA and DNA standards * dual-labeled sequence-specific probes

63 Epigenetics Application Webpage For more information visit us at: