epi tect methylation qpcr arrays 2013

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Sample & Assay Technologies

EpiTect Methyl II PCR Array System

Samuel J. Rulli, Jr., Ph. D.Epigenetics Applications Scientist [email protected]@QIAGEN.com

The EpiTect Methyl qPCR Arrays and Assays are intended for molecular biology applications.This product is not intended for diagnosis, prevention, or treatment of disease.

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The EpiTect Methyl II PCR Arrays and Assays are intended for molecular biology applications.This product is not intended for diagnosis, prevention, or treatment of disease.

Questions, Comments, Concerns?US Applications Support

[email protected]

Questions, Comments, Concerns?Global Applications Support

[email protected]

EpiTect Methyl II PCR Array System

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Disclaimer and intended useEpiTect Methyl II PCR Array product line

EpiTect® Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

The EpiTect Methyl II qPCR system enables pathway or disease-focused profiling of regional DNA methylation using MethylScreen™ technology provided under license from Orion Genomics, LLC.

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.

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Topics for Discussion

� Introduction� DNA Methylation Overview� Challenges & Solutions

� The EpiTect Methyl II PCR Array System� Principle and Protocol

� Research Applications� Pyrosequencing� Summary

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Basic Gene Expression Model

Structural Gene “A”

mRNA ”A”

Protein “A”

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The Current Model

Structural Gene “A”or Reporter systemNFκB BS

Activated Transcription Factors

p53 BS

p53

NFκB

Protein “A”

mRNA ”A”

MeMeMe

Histones

Histone-DNA Interactions

DNA Methylation

Me AcMe Me MeMe

DNA Methylation

RNAi:shRNAmiRNA

Transcription Initiation Complex

+

–

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Structural Gene “A”or Reporter systemTSS (+1)

+

–

Activated Transcription Factors

Transcription Initiation Complex

p53 BS NFκB BS

p53

NFκB

Histones

Histone-DNA Interactions

Me AcMeMe

Protein “A”

mRNA ”A”

DNA Methylation

Me MeMeMe Me

DNA Methylation

RNAi:shRNAmiRNA

The Current Model

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� Stable, covalent DNA modification that targets CpG dinucleotides

� 70%-80% of the CpG dinucleotides are methylated in healthy mammalian cells

� These methylated regions are typical of the bulk chromatin that is relatively inaccessible to transcription factors.

� Associated with gene silencing

DNA Methylation: Introduction

cytosine

N

N

NH2

ON

N

NH2

O

R

H

S-Enz

H

CH3

N

N

NH2

O CH3+DNMT -DNMT

AdoMet AdoHcy

5-methyl-cytosine

Adapted from Herman, J. and Baylin, S. N. Engl. J. Med. 2003

1111 2222 3333

1111 2222 3333

Normal

Cancer

Promoter Region

Expression

No Expression

� CpG islands: CpG-rich regions in human gene promoters

� Promoter CpG islands are usually unmethylated in normal tissues.

� Exceptions include genes involved in X-chromosome inactivation, gene imprinting and tissue-specific expression.

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DNA Methylation and Transcriptional Repression

Blood, 93:4059-4070, 1999

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Critical Biological Roles for DNA Methylation

� Roles in normal development and disease progression

� Potentially silences expression of critical tumor s uppressors

� Involves all cellular pathways

� Methylated DNA can be detected in heterogenous flui ds

� Breast cancer: nipple aspirate

� Lung cancer: bronchoalveolar lavage

� Prostate cancer: urine

� Multiple cancers: plasma and serum

DNA Methylation: Promising Biomarker

� Early detection

� Predicting prognosis

� Mechanism of Action for Therapy Resistance/ Therapy Choice

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Challenges in Existing Technologies

� Current Workflow for DNA Methylation Analysis:

� Commonly used pretreatment – sodium bisulfite conversion

� Methods of analysis – Pyrosequencing, Methylation-specific PCR (MSP), bisulfite sequencing, next-generation sequencing, MALDI-TOF MS, etc.

� Benefits – Single-base resolution of methylome

� Drawbacks – Protocol Optimization, laborious for many samples, need for specialized equipment

�WHY EpiTect Methyl II qPCR System?�Available for any laboratory with qPCR instrument�Complete System- Sample Isolation to DATA Analysis�Regional Methylation Pattern �More Biology - Less Time�Complimentary to Bisulfite Based Methods

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Site Specific DNA Methylation AnalysisUsing Bisulfite Treatment

G T CAG T GAmCG

G T UAG T GA CG

G T T AG T GA CG

Bisulfite Conversion

PCR

G T C AG T GA CG

G T U AG T GA UG

G T T AG T GA T G


PCR

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Challenges in Existing Technologies

� Current Workflow for DNA Methylation Analysis:

� Commonly used pretreatment – sodium bisulfite conversion

� Methods of analysis – Pyrosequencing, Methylation-specific PCR (MSP), bisulfite sequencing, next-generation sequencing, MALDI-TOF MS, etc.

� Benefits – Single-base resolution of methylome

� Drawbacks – Protocol Optimization, laborious for many samples, need for specialized equipment

�WHY EpiTect Methyl II qPCR System?�Available for any laboratory with qPCR instrument�Complete System- Sample Isolation to DATA Analysis�Regional Methylation Pattern �More Biology - Less Time�Complimentary to Bisulfite Based Methods

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EpiTect DNA Methylation PCR SystemComplete Solution from Sample Isolation through Dat a Analysis

� DNA Isolation: QIAGEN Blood and Tissue Kit

� EpiTect Methyl II DNA Restriction Kit� For fresh/frozen tissues and cells

� EpiTect Methyl II PCR Arrays� 96 or 384 well format compatible with most qPCR instruments

� Cataloged - multiple assays (24 or 96) on one PCR Plate

� Custom - available for every promoter in the human, mouse, and rat genome

� EpiTect Methyl II PCR Assays� Primers for every promoter in the human, mouse, or rat genome with CpG island

� EpiTect qPCR primer assay search portal

� Optimized RT 2 SYBR Green Master Mix

� EpiTect Methyl II Data Analysis (Excel template)

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DNA Methylation PCR Array Protocol Overview

(0.5 – 4 ug of Genomic DNA)

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Sample Preparation:QIAGEN DNeasy Blood and Tissue Kit

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How It Works: Principle behind EpiTect Methyl II PCR Array

� Based on the quantitative detection of remaining ta rget DNA molecules after treatment with a Methylation-sensitive (MSRE) and a Methylation-dependent (MDRE) restriction enzyme mixtures.

Enzyme How It Works Remaining DNA

MOCK No enzyme added Input fraction

MSRE Digests unmethylated and partially

methylated DNA copies Methylated fraction

MDRE Digests methylated and partially

methylated DNA copies Unmethylated fraction

MSRE & MDRE (Double)

Digests unmethylated, partially methylated, and methylated DNA copies

Fraction resistant to enzyme digestion (Analytical window)

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Which EpiTect Methyl II PCR Array do I use?


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“Catalogued” EpiTect Methyl II PCR Arrays

Cancer Related

� Breast Cancer

� Epithelial to Mesenchymal Transition

� Gastric Cancer

� Liver Cancer

� Lung Cancer

� Prostate Cancer

� Colon Cancer

� Leukemia & Lymphoma

� Tumor Suppressor Genes

� Melanoma

� Cancer miRNAs

Simultaneously analyze the DNA methylation status of 22- or 94-gene panels associated with specific cancer types, other diseases or biological pathways.

Pathway-Focused Methyl qPCR Arrays

� Apoptosis

� DNA Repair

� Stem Cell Transcription Factors

� Inflammatory Response

� T Cell Activation

� Cytokine Production


� Homeobox (HOX) Genes

� Polycomb (PcG) Genes

� Cell Cycle

� Toll-Like Receptor Signaling

� TGF-Beta/BMP Signaling

� Wnt Signaling

96 and 384 well qPCR Arrays-Human and Mouse

Custom Arrays and Custom Assays Are AvailableAvailable for most CpG island-containing promoter in the human, mouse, or rat genome.

http://www.sabiosciences.com/dna_methylation_custom_PCRarray.php

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Data Analysis


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EpiTect Data Analysis

�Excel Analysis Templates

� Quickly Understand the Biology:Results automatically calculated, analyzed, and annotated

�No Software to download, license or install:Simply paste in your raw Ct values

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Advantages of the EpiTect Methyl II PCR System

� Single Gene, Pathway or Disease-Focused Analysis� Screen large number of samples for few genes with single

assays� Simultaneously detect the methylation status of 22-94 genes� Compliments & validates genome-wide methylation studies

� Fast, Reliable & Sensitive� Direct measurement of methylated & unmethylated DNA� Similar results and sensitivity as bisulfite sequencing, with no

bisulfite conversion� Higher throughput than bisulfite-PCR� Highly reproducible

� Genome-Wide Coverage� Primers detecting methylation status of any human, mouse, or

rat gene with predicted CpG island� Bench-validated qPCR primers

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Four selective methylation-specific digestion restriction

qPCR results

Unmethylated

Hypermethylated

Unmethylated

Hypermethylated

Unmethylated

EpiTect Methyl II PCR Array Principle:Representational design

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Four selective methylation-specific digestion restriction

qPCR results

Unmethylated

Hypermethylated

Unmethylated

Hypermethylated

Unmethylated1111Sample 1

methylated1111

Sample 2

� Each CpG Island is interrogated at multiple sites f or both RE Digests� Unbiased qPCR since both primers are the same (vali d ∆∆∆∆Ct calculations)� Internal Standard for each Assay (mock digest versu s total digest)� Representational Design

EpiTect Methyl II PCR Array Principle:Representational design

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� Targets� Primers available for approximately 13,000 promoter CpG islands� Design includes CpG island or CpG dense areas associated with a transcription

start site (TSS)� CpG island & TSS definitions: UCSC Genome Bioinformatics Site & NCBI� Each assay corresponds to one distinct CpG island in a promoter region� EpiTect Methyl qPCR Assay Search Portal:

http://www.sabiosciences.com/dna_methylation_qPCRpr imer.php

� Amplicon Specifics� Selected from 5 kb upstream to 3 kb downstream of TSS� Each includes at least three or more of both MSRE and MDRE restriction sites� Size: 150 to 400 bp (240 bp average)

EpiTect Methyl II PCR Assays

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Laboratory QC Validation of ALL EpiTect Methyl II PCR Assays & Arrays

All EpiTect Methyl II PCR Arrays and Assays are laboratory validated:

� Assays in each PCR Array are tested for

�Sensitivity:�Constant C(t) value for same amount of template

�Amplification Efficiency:�Single curve analysis and standard curve dilutions

�Specificity:�Single melt-peak during dissociation

�Validation is done on every lot of primers and every EpiTect Methyl II PCR Array�Performance of all EpiTect Methyl II PCR Assays is guaranteed when used with the RT2 SYBR-Green qPCR Mastermix

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EpiTect Methyl qPCR Assay Efficiency

0%

20%

40%

60%

80%

100%

120%

140%

0 100 200 300 400 500

PC

R E

ffici

ency

Amplicon length in bp

Mouse

Human

Amplification Efficiency Amplification and Dissociation Curves

�Unique qPCR cycling Conditions (critical for assay performance)

�Validated Primer Specificity & High Amplification Efficiency

�Similar Amplification Efficiencies for Each Primer Pair

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Melting Curve Analysis

� Example of QC criteria for every PCR Assay manufactured by SABiosciences

� Single peak dissociation curves

� Single gel bands of predicted size

Single Dissociation Peaks for Every Gene Assay

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EpiTect Methyl II PCR Arrays Comparable to Bisulfite Sequencing

EpiTect Methyl II PCR Assays generated quantitative methylation data comparable to bisulfite sequencing data.

Unmethylated CpG sitesMethylated CpG sites

CDH13

MCF7 MB231 HeLa

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EpiTect Methyl qPCR Assay Sensitivity

Methylated tumor DNA detectable in heterogeneous samples containing as little as 5% tumor.

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

100.0 75.0 50.0 33.3 12.5 6.3 0.0

PE

RC

EN

TA

GE

OF

TO

TA

L IN

PU

T D

NA

PERCENT SKBR3 GENOMIC DNA (%)

SENSITIVITY COMPARABLE TO BISULFITE SEQUENCING

Unmethylated

Methylated

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EpiTect Methyl II PCR Arrays Comparable to Illumina Infinium BeadChip

�Comparison between 94 CpG sites (22 shown) important for breast cancers in MCF-7 cells�Similar results between 2 different platforms using different preparation methods�Methyl II PCR assays can be used to validate results from large scale screening projects

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Research Applications:Methylation in Breast Cancer

Obtained genomic DNA from Human Breast Cancer Cell lines (ATCC) Normal Breast Tissue (Capital Bioscience)

Is there a difference in the Methylation status of breast cancer associated genes

among commonly used Breast Cancer Cell lines?

EpiTect Methyl II Breast Cancer PCR Array

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Human Breast Cancer EpiTect Methyl II Complete PCR Array: Functional gene groupings

�Cell Cycle, Growth, Differentiation & Development: APC, ATM, BIRC5, BRCA1, BRCA2, CCNA1, CCND2, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CHFR, GADD45A, GPC3, HOXD11, MEN1, PALB2, RASSF1, SFN (14-3- 3 sigma), TERT, TGFB2, TGFBI, TGFBR1, TP73, TWIST1, VHL, WIF1, WT1, WWOX.

�Transcription Factors: APC, BRCA1, BRCA2, MYOD1, CDKN2A, CDX2, HIC1, HOXA5, HOXD11, ID4, MSX1, PAX5, PLAGL1, PRDM2, PROX1, RB1, RUNX3, TP73, TWIST1, VHL, WT1, ZMYND10.

�DNA Damage Repair: APC, BRCA1, BRCA2, GADD45A, MLH1, RASSF1, TERT, VHL, WWOX.

�Apoptosis & Anti-Apoptosis: APC, BIRC5, BRCA1, BRCA2, DAPK1, MEN1, PYCARD, TNFRSF10C, TNFRSF10D, TP73, VHL, WWOX.

�Cell Adhesion: APC, CDH1, CDH13, CADM1, CLSTN1, DSC3, EPCAM, JUP, THBS1.

�Growth Factors: BMP6, IGFBP7, IGFBPL1, WIF1.

�Chemokine & Cytokine Signaling: CXCL12, TNFRSF10C, TNFRSF10D.

�Circadian Rhythm: PER1, PER2.

�Solute Carrier Family: SLC5A8.

�Drug Metabolism: CYP1B1, GSTP1.

�Fatty Acid Metabolism: HSD17B4.

�Heparan Sulfate Biosynthetic Enzymes: HS3ST2, HS3ST3B1.

�Extracellular Matrix: LOX, MUC2, SLIT2, SLIT3, TGFBI, THBS1, TIMP3, WIF1.

�Protein Kinase & Protein Kinase Adaptor Proteins: PRKCDBP.

�Phosphatases: PTEN.

�Prostaglandin-Endoperoxide Synthase: PTGS2.

�DNA Methylation: MEN1, MGMT, PRDM2.

�Proteases & Inhibitors: ADAM23, CST6, CTSZ, KLK10, THBS1, TIMP3.

�Caveolae Protein: CAV1.

�Nuclear Receptors: ESR1, PGR, RARB, RBP1.

�Hormones & Receptors: ESR1, PGR.

�Histidine Triad Gene Family: FHIT.

�Ras Associated Protein Superfamily: RRAD, RASSF1.

�WNT Signaling Protein: SFRP1, SFRP2, WIF1.

�Others: CALCA, EPB41L3, PDLIM4, RARRES1.

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Genomic DNA from 10 breast cancer, 1 prostate cancer cell line and normal human breast tissue was isolated and analyzed with the EpiTect Human Breast Cancer Complete Panel PCR Array. The heat map compares the methylation levels of the 94 genes on the array.

EpiTect Biomarker Discovery

CONCLUSION: EpiTect Methyl II PCR Arrays can be effectively used to identify novel methylated biomarkers.

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What’s next?

Verify gene expressionUsing RT2 Profiler PCR Arrays

Focus on individual CpG sites by pyrosequencing

EpiTect Biomarker Discovery

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Site Specific DNA Methylation AnalysisUsing Bisulfite Treatment

� Importance

� Crucial step to analyze CpG methylation

� Required to distinguish between methylated and

unmethylated cytosines

GG T C AG T GA CG

GG T U AG T GA UG

GG T T AG T GA T G

GG T C AG T GAmCG

GG T U AG T GA CG

GG T T AG T GA CG


PCR


PCRCells

Blood

Biopsies

Paraffinslides

gDNAConverted

DNA

Bisulfite treatmentconverts

unmethylated C into U

Analysis

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Assay Design Sample prep Pyrosequencing

~ 2h ~ 10-60 min~ 15 min

PCR

Region of interest amplified with a biotinylated primer

Separation to single stranded DNA using streptavidin-coated beads.

Annealing of sequencing primer.

Sequencing-by-synthesis. Sequence data generated from the first base next to the sequencing primer.

Sequence context as built in control

The Principle of Pyrosequencing TechnologyWorkflow

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PyroMark Q24 PyroMark Q96 ID PyroMark Q96 MD PyroMark Q96 MD Automated

Throughput 1–24 samples 1–96 samples 1–96 samples 10–960 with aut omation option

Running volume 25 µl 40 µl 12 µl 12 µl

PCR requirements

10–20 µl (~1-3 pmol of product)

20–40 µl(2-4 pmol of product)

5–10 µl (0.5-1.5 pmol of product)

5–10 µl (0.5-1.5 pmol of product)

Read lengths(estimates)

SQA ~50 – 100 bpSNP ~10 – 100 bpAQ ~10 – 100 bpCpG ~10 – 120 bp

SQA ~40 – 70 bpSNP ~10 – 100 bpAQ ~10 – 100 bpCpG ~10 – 120 bp

SNP ~10 – 100 bpAQ ~10 – 100 bpCpG ~10 – 140 bp

SNP ~10 – 100 bpAQ ~10 – 100 bpCpG ~10 – 140 bp

Main applications

Genetic testingEpigeneticsMicrobilogy

Genetic testingEpigenetics Microbiology

EpigeneticsGenetic testing

EpigeneticsGenetic testing(SNP/AQ only in batch mode)

Sensitivity 5% mutation95% wt

10% mutation90% wt

2% mutation98% wt

2% mutation98% wt

Instrument overviewSoftware functionality and application areas

** Additional Software (SW) (PyroMark CpG SW) needed on ID and MD instruments

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Pyrosequencing for DNA Methylation AnalysisPyroMark CpG Assays – Release Information

PyroMark CpG AssaysPre-designed DNA-methylation assays

PyroMark CpG Assays

� Human � Number of assays: over 30,000� Number of CpG Islands with assay: ~12,000

(~80%)

� Mouse � Number of assays: over 30,000� Number of CpG Islands with assay: ~11,000

(<80%)

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Pyrosequencing WorkflowPCR

� Can Use Any PCR Machine / PCR Reagents / Compatible with Q-PCR� Amplify relevant region by PCR (60 - 1000 bp)

� Can Use Very Short PCR Products if Desired (i.e. Degraded DNA)� One primer is biotinylated

PCR primer

Region of interestPCR primerbiotinylated

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Pyrosequencing WorkflowSample preparation

� Immobilize biotinylated PCR products onto streptavidin coated beads

� Separate strands by denaturation in NaOH

� Wash/neutralize the immobilized strand

� Anneal sequencing primer

Assay Design PCR PyrosequencingSample prep

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Pyrosequencing for DNA Methylation Analysis Analyzing a Pyrogram for DNA-methylation

� Sequence to be analyzed:

� After bisulfite conversion:

� Biotinylated PCR strand:

• A G T T A C G A C

• A G T T A C G A C • A G T T A Cm

G A C • and

• A G T T A T G A T • A G T T A C G A T • and

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Pyrosequencing for DNA Methylation Analysis Analyzing a Pyrogram for DNA-methylation

� Sequence to be analyzed:

� After bisulfite conversion:

� Biotinylated PCR strand:

� Analyzed sequence:CpG methylation level:

• A G T T A C G A C

• A G T T A C G A C • A G T T A Cm

G A C • and

• A G T T A T G A T • A G T T A C G A T • and

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Pyrosequencing for DNA Methylation AnalysisA Range of Analysis Possibilities

� One gene at a time� Several genes in the same analysis (analyze up to 96 different assays in one run)� Global methylation level

� Estimate the global methylation levels using repetitive elements

57%

EE SS AA TT GG AA TT

5

CC GG TT GG

0

50

100

150

200

A4: TAYGGTTTGTA

� Multiple consecutive CpG sites

� Any single CpG site

37% 35% 36% 33% 33% 32% 33% 30% 31% 38% 37% 29% 36% 30% 31% 34% 34%

EESS AATTCCGGTT

5

AATTGGTTGG

10

AATTCCGGAA

15

TTTTCCTTGG

20

TTCCGGTTGG

25

TTAATTCCTT

30

GGAATTCCGG

35

GGAATTAATT

40

TTGGTTCCGG

45

AATTCCAAGG

50

TTCCTTGGTT

55

CCAAGGTTCC

60

GGAATTAATT

65

CCTTGGAATT

70

CCGGAAGGAA

75

TTGGAA TTTT

80

CCTTGGTTCC

85

AAGGTTCCTT

90

GGTTCCGGAA

95

TTAATTCCGG

100

AATTCCAAGG

105

TTCCGGGGTT

110

AATTCCAAGG

115

TTTTCCGGAA

120

TT

0

500

1000

1500

2000

B 7 : Y G G T A T G T G G A T A T T T T Y G Y G T G G G T A T T T T T AYGGGGAT AT T T T GGT T AT YGT YGYGYGGAT AT T T T T AYGAGGAT AT T T YGGT YGYGYGGAT AT T T AT YGYGGGGAYGT T T YGAT T T T AT T T T AT T T GT T G

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DNA Methylation Analysis at QIAGENComplete Solution from Sample Isolation through Dat a Analysis

�DNA Methylation is a promising biomarker and is important in regulating gene expression through several mechanisms

�DNA Methylation can be identified using simple restriction digests on a regional basis using the EpiTect Methyl II PCR Array System

�Complete system from DNA isolation through data analysis

�DNA Methylation can be identified on site-by-site basis using EpiTect pyrosequencing

�Simple, easy to use system with predesigned assays

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EpiTect Methyl II PCR System

�Pilot Project Starter Offer:�2 Free EpiTect Methyl II Signature Panels or 2 Free EpiTectMethyl II Assays

�With purchase of:�DNA RE Kit�SYBR Green MasterMix

�Call 1-888-503-3187

�Use Code: FDK-MTFAS22Outside US and Canada? Request demo [email protected]

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Cancer Related

� Breast Cancer

� Epithelial to Mesenchymal Transition

� Gastric Cancer

� Liver Cancer

� Lung Cancer

� Prostate Cancer

� Colon Cancer

� Leukemia & Lymphoma


� Melanoma

� Cancer miRNAs

Pathway-Focused Methyl qPCR Arrays

� Apoptosis

� DNA Repair

� Stem Cell Transcription Factors

� Inflammatory Response

� T Cell Activation

� Cytokine Production


� Homeobox (HOX) Genes

� Polycomb (PcG) Genes

� Cell Cycle

� Toll-Like Receptor Signaling

� TGF-Beta/BMP Signaling

� Wnt Signaling

96 and 384 well qPCR Arrays-Human and Mouse

EpiTect Methyl II Pilot Study

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EpiTect Methyl qPCR System

The EpiTect Methyl qPCR Arrays and Assays are intended for molecular biology applications.This product is not intended for diagnosis, prevention, or treatment of disease.

Questions, Comments, Concerns?US Applications Support

[email protected]

Questions, Comments, Concerns?Global Applications Support

[email protected]

epi tect methylation qpcr arrays 2013

Documents

ugpcrg t t ag t g

cgpcrg t t ag t g

dna methylation roles

cgg t u ag t g

cgg t c ag t g

serumdna methylation

intended use epitect

methylationspecific