Epi tect methylation qpcr arrays 2013

EpiTect Methyl II PCR Array System

Samuel J. Rulli, Jr., Ph. D.
Epigenetics Applications Scientist
Samuel.Rulli@QIAGEN.com
QIAwebinars@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.
1
Sample & Assay Technologies

EpiTect Methyl II PCR Array System

Questions, Comments, Concerns?
US Applications Support
888-503-3187
support@sabiosciences.com
̣

Global Applications Support
SAB.EU@qiagen.com

The EpiTect Methyl II PCR Arrays and Assays are intended for molecular biology applications.
2

Disclaimer and intended use
EpiTect 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 productspecific 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.
̣

3

Topics for Discussion
Introduction
DNA Methylation Overview
Challenges & Solutions

The EpiTect Methyl II PCR Array System
Principle and Protocol

Research Applications
Pyrosequencing
Summary

4

Basic Gene Expression Model

Protein “A”

mRNA ”A”

Structural Gene “A”

5

The Current Model
Activated
Transcription Factors

RNAi:
shRNA
miRNA

Protein “A”
NFκB

+

p53

Transcription
Initiation Complex

mRNA ”A”

–
Histones
p53 BS Me

Me

Me

Me Me

NFκB BS

DNA Methylation

Histone-DNA
Interactions

Ac

Me

Me Me

or Reporter system

DNA Methylation

6

The Current Model
Activated
Transcription Factors

RNAi:
shRNA
miRNA

Protein “A”
NFκB

+

p53

Transcription
Initiation Complex

mRNA ”A”

–
Histones
p53 BS Me

Me

Me

Me Me

NFκB BS

DNA Methylation

Histone-DNA
Interactions

Ac

Me

Me Me

TSS (+1) or Reporter system

DNA Methylation

7

DNA Methylation: Introduction
NH2
N
O
N

H NH2
H
+DNMT
O
CH3
N S-Enz
AdoMet AdoHcy R
N

NH2
-DNMT

N
CH3

O
N

5-methyl-cytosine

cytosine

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

1

Normal

2

3

Expression

Promoter
Region

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

1

2

3

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.

8

DNA Methylation and Transcriptional Repression

9

Blood, 93:4059-4070, 1999


Critical Biological Roles for DNA Methylation
Roles in normal development and disease progression
Potentially silences expression of critical tumor suppressors
Involves all cellular pathways
Methylated DNA can be detected in heterogenous fluids
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

10

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
11

Site Specific DNA Methylation Analysis
Using Bisulfite Treatment
m

G T C AG T G A CG

G T C AG T G A CG

Bisulfite Conversion


G T U AG T G A CG

G T U AG T G A UG

PCR

G T T AG T G A CG

PCR

G T T AG T G A T G

12

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
13

Introduction


Pyrosequencing
Summary

14

EpiTect DNA Methylation PCR System
Complete Solution from Sample Isolation through Data 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 RT2 SYBR Green Master Mix
EpiTect Methyl II Data Analysis (Excel template)
15

DNA Methylation PCR Array Protocol Overview
(0.5 – 4 ug of
Genomic DNA)

16


Sample Preparation:
QIAGEN DNeasy Blood and Tissue Kit

17

(0.5 – 4 ug of
Genomic DNA)

18

How It Works:
Principle behind EpiTect Methyl II PCR Array
Based on the quantitative detection of remaining target DNA molecules
after treatment with a Methylation-sensitive (MSRE) and a Methylationdependent (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)

19

(0.5 – 4 ug of
Genomic DNA)

20

(0.5 – 4 ug of
Genomic DNA)

Which EpiTect Methyl II
PCR Array do I use?

21

“Catalogued” EpiTect Methyl II PCR Arrays
Simultaneously analyze the DNA methylation status of 22- or 94-gene panels associated with
specific cancer types, other diseases or biological pathways.
96 and 384 well qPCR Arrays-Human and Mouse
Cancer Related

Pathway-Focused Methyl qPCR Arrays

Breast Cancer

Apoptosis

Epithelial to Mesenchymal
Transition

DNA Repair

Gastric Cancer

Inflammatory Response

Liver Cancer

T Cell Activation

Lung Cancer

Cytokine Production

Prostate Cancer

Tumor Suppressor Genes

Colon Cancer

Homeobox (HOX) Genes

Leukemia & Lymphoma

Polycomb (PcG) Genes


Cell Cycle

Melanoma

Toll-Like Receptor Signaling

Cancer miRNAs

Stem Cell Transcription Factors

TGF-Beta/BMP Signaling
Wnt Signaling

Custom Arrays and Custom Assays Are Available
Available for most CpG island-containing promoter in the human, mouse, or rat genome.
22

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

(0.5 – 4 ug of
Genomic DNA)

Data
Analysis

23

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

24

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
25

EpiTect Methyl II PCR Array Principle:
Representational design

Unmethylated

Unmethylated

Hypermethylated

qPCR results

Four selective methylation-specific digestion restriction

26

EpiTect Methyl II PCR Array Principle:
Representational design

Sample 1

Sample 2

Unmethylated
1

Unmethylated

Hypermethylated

methylated
1

qPCR results

Each CpG Island is interrogated at multiple sites for both RE Digests
Unbiased qPCR since both primers are the same (valid ∆Ct calculations)
Internal Standard for each Assay (mock digest versus total digest)
Representational Design

Four selective methylation-specific digestion restriction

27

EpiTect Methyl II PCR Assays
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_qPCRprimer.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)
28

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
29

EpiTect Methyl qPCR Assay Efficiency
Amplification Efficiency

Amplification and Dissociation Curves

140%

PCR Efficiency

120%
100%
80%
60%

Mouse

40%

Human

20%
0%
0

100

200

300

400

500

Amplicon length in bp

Unique qPCR cycling Conditions (critical for assay performance)
Validated Primer Specificity & High Amplification Efficiency
Similar Amplification Efficiencies for Each Primer Pair

30

Melting Curve Analysis
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

31


every PCR Assay
manufactured by
SABiosciences
curves
Single gel bands of
predicted size

32


every PCR Assay
manufactured by
SABiosciences
curves
Single gel bands of
predicted size

33

Comparable to Bisulfite Sequencing
CDH13

MCF7

MB231

HeLa

Unmethylated CpG sites
Methylated CpG sites

EpiTect Methyl II PCR Assays generated quantitative
methylation data comparable to bisulfite sequencing data.
34

EpiTect Methyl qPCR Assay Sensitivity

PERCENTAGE OF TOTAL INPUT DNA

SENSITIVITY COMPARABLE TO BISULFITE SEQUENCING
100.00%
90.00%
80.00%
70.00%
60.00%
50.00%

Unmethylated

40.00%

Methylated

30.00%
20.00%
10.00%
0.00%
100.0

75.0
50.0
33.3
12.5
6.3
PERCENT SKBR3 GENOMIC DNA (%)

0.0

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

35

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
36

Introduction


Pyrosequencing
Summary

37

Research Applications:
Methylation in Breast Cancer
Is there a difference in the Methylation status of breast cancer associated genes
among commonly used Breast Cancer Cell lines?

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

EpiTect Methyl II Breast Cancer PCR Array

38

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.

39

Others: CALCA, EPB41L3, PDLIM4, RARRES1.


EpiTect Biomarker Discovery
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.

CONCLUSION:
can be effectively used to
identify novel methylated
biomarkers.

40

EpiTect Biomarker Discovery
What’s next?
Verify gene expression
Using RT2 Profiler
PCR Arrays
Focus on individual
CpG sites by
pyrosequencing

41

Introduction


Pyrosequencing
Summary

42

Site Specific DNA Methylation Analysis
Using Bisulfite Treatment
Importance

m

GG T C AG T G A CG

Crucial step to analyze CpG methylation
Required to distinguish between methylated and


GG T U AG T G A CG
PCR

unmethylated cytosines

GG T T AG T G A CG
GG T C AG T G A CG

GG T U AG T G A UG
Cells
Blood

PCR

Bisulfite treatment
converts
unmethylated
C into U

gDNA

Converted
DNA

GG T T AG T G A T G

Analysis

Biopsies

Paraffin
slides
43

The Principle of Pyrosequencing Technology
Workflow

~ 2h

Assay Design

PCR
Region of interest
amplified with a
biotinylated primer

~ 15 min

Sample prep

~ 10-60 min

Pyrosequencing

Separation to single
stranded DNA using
streptavidin-coated
beads.

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

Annealing of sequencing
primer.

Sequence context as built in
control

44

Instrument overview
Software functionality and application areas
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 automation
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
SNP
AQ
CpG

SQA
SNP
AQ
CpG

SNP
AQ
CpG

SNP
AQ
CpG

Main
applications

Genetic testing
Epigenetics
Microbilogy

Genetic testing
Epigenetics
Microbiology

Epigenetics
Genetic testing

Epigenetics
Genetic testing
(SNP/AQ only in batch
mode)

Sensitivity

5% mutation
95% wt

10% mutation
90% wt

2% mutation
98% wt

2% mutation
98% wt

~50 – 100 bp
~10 – 100 bp
~10 – 100 bp
~10 – 120 bp

~40 – 70 bp
~10 – 100 bp
~10 – 100 bp
~10 – 120 bp

~10 – 100 bp
~10 – 100 bp
~10 – 140 bp

~10 – 100 bp
~10 – 100 bp
~10 – 140 bp

45
** Additional Software (SW) (PyroMark CpG SW) needed on ID and MD instruments


Pyrosequencing for DNA Methylation Analysis
PyroMark CpG Assays – Release Information
PyroMark CpG Assays
PyroMark CpG Assays
Pre-designed
DNA-methylation 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%)

46

Pyrosequencing Workflow
PCR
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 interest
PCR primer
biotinylated

47

Pyrosequencing Workflow
Sample preparation
Assay Design

PCR

Sample prep

Pyrosequencing

Immobilize biotinylated PCR
products onto streptavidin coated beads
Separate strands by denaturation in NaOH
Wash/neutralize the immobilized strand
Anneal sequencing primer

48

Analyzing a Pyrogram for DNA-methylation
Sequence to be analyzed:

•

AGTTAC GAC
m

•
After bisulfite
conversion:

AG T TAC GAC•

and A G T T A C G A C
•

•

AG TTATGAT •

and A G T T A C G A T
•

Biotinylated PCR strand:

49

Analyzing a Pyrogram for DNA-methylation
Sequence to be analyzed:

•

AGTTAC GAC
m

•
After bisulfite
conversion:

AG T TAC GAC•

and A G T T A C G A C
•

•

AG TTATGAT •

and A G T T A C G A T
•

Biotinylated PCR strand:
Analyzed sequence:
CpG methylation level:

50

A Range of Analysis Possibilities
Any single CpG site
A4: TAYGGTTTGTA
57%
200
150
100
50
0
E

S

A

T

G

A

T

C

G

T

G

5

Multiple consecutive CpG sites

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 YGYGGGGAY
37%

35%

36%

33%

33%

32%

33%

30%

31%

38%

37%

29%

36%

30%

31%

34%

34%

2000
1500
1000
500
0
E S A TCGT A TGTGA TCGA T TC TG TCGTGTA TC TGA TC GGA TA T TGTCGA TCA GTC TGTCAGTCGA T A TC TGA TCGAGA TGA T TC TGTCAGTC TGT CGA TA TCGA TCAGTC GGTA TCAGT TCGA T
5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

115

120

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
51

Introduction


Pyrosequencing
Summary

52

DNA Methylation Analysis at QIAGEN
Complete Solution from Sample Isolation through Data 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

53

EpiTect Methyl II PCR System

Pilot Project Starter Offer:
2 Free EpiTect Methyl II Signature Panels or 2 Free EpiTect
Methyl II Assays
With purchase of:
DNA RE Kit
SYBR Green MasterMix
Call 1-888-503-3187
Use Code: FDK-MTFAS22

Outside US and Canada?
Request demo QIAwebinars@QIAGEN.com
54

EpiTect Methyl II Pilot Study
96 and 384 well qPCR Arrays-Human and Mouse
Cancer Related

Pathway-Focused Methyl qPCR Arrays

Breast Cancer

Apoptosis

Epithelial to Mesenchymal
Transition

DNA Repair

Gastric Cancer

Inflammatory Response

Liver Cancer

T Cell Activation

Lung Cancer

Cytokine Production

Prostate Cancer


Colon Cancer

Homeobox (HOX) Genes

Leukemia & Lymphoma

Polycomb (PcG) Genes


Cell Cycle

Melanoma

Toll-Like Receptor Signaling

Cancer miRNAs

Stem Cell Transcription Factors

TGF-Beta/BMP Signaling
Wnt Signaling

55

EpiTect Methyl qPCR System

US Applications Support
888-503-3187
support@sabiosciences.com
̣

Global Applications Support
SAB.EU@qiagen.com

The EpiTect Methyl qPCR Arrays and Assays are intended for molecular biology applications.
56

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