1 Sample & Assay Technologies EpiTect Methyl II PCR Array System Samuel J. Rulli, Jr., Ph. D. Epigenetics Applications Scientist [email protected][email protected]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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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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Sample & Assay Technologies
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
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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Sample & Assay Technologies
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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Sample & Assay Technologies
Basic Gene Expression Model
Structural Gene “A”
mRNA ”A”
Protein “A”
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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
� 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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Sample & Assay Technologies
DNA Methylation and Transcriptional Repression
Blood, 93:4059-4070, 1999
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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
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
Bisulfite Conversion
PCR
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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
DNA Methylation PCR Array Protocol Overview
(0.5 – 4 ug of Genomic DNA)
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Sample & Assay Technologies
DNA Methylation PCR Array Protocol Overview
Sample Preparation:QIAGEN DNeasy Blood and Tissue Kit
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Sample & Assay Technologies
DNA Methylation PCR Array Protocol Overview
(0.5 – 4 ug of Genomic DNA)
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Sample & Assay Technologies
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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Sample & Assay Technologies
DNA Methylation PCR Array Protocol Overview
(0.5 – 4 ug of Genomic DNA)
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Sample & Assay Technologies
DNA Methylation PCR Array Protocol Overview
Which EpiTect Methyl II PCR Array do I use?
(0.5 – 4 ug of Genomic DNA)
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Sample & Assay Technologies
“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
� Tumor Suppressor Genes
� 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.
� 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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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
� 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:
� 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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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
Single Dissociation Peaks for Every Gene Assay
� Example of QC criteria for every PCR Assay manufactured by SABiosciences
� Single peak dissociation curves
� Single gel bands of predicted size
Melting Curve Analysis
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Sample & Assay Technologies
Single Dissociation Peaks for Every Gene Assay
� Example of QC criteria for every PCR Assay manufactured by SABiosciences
� Single peak dissociation curves
� Single gel bands of predicted size
Melting Curve Analysis
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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
Human Breast Cancer EpiTect Methyl II Complete PCR Array: Functional gene groupings
�Ras Associated Protein Superfamily: RRAD, RASSF1.
�WNT Signaling Protein: SFRP1, SFRP2, WIF1.
�Others: CALCA, EPB41L3, PDLIM4, RARRES1.
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Sample & Assay Technologies
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.
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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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
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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Sample & Assay Technologies
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
Pathway-Focused Methyl qPCR Arrays
� Apoptosis
� DNA Repair
� Stem Cell Transcription Factors
� Inflammatory Response
� T Cell Activation
� Cytokine Production
� Tumor Suppressor Genes
� 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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Sample & Assay Technologies
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