1. EpiTect ChIP qPCR System
A Complete System from Chromatin
ImmunoPrecipitation to Data Analysis
Samuel Rulli, Ph.D.
Applications Scientist
Samuel.Rulli@QIAGEN.com
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Sample & Assay Technologies
2. EpiTect ChIP qPCR System
A Complete System from Chromatin
ImmunoPrecipitation to Data Analysis
Questions, Comments, Concerns?
US Applications Support
Questions, Comments, Concerns?
Global Applications Support
SABio@qiagen.com
888-503-3187
̣
support@sabiosciences.com
*
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Sample & Assay Technologies
4. Webinar Overview
Introduction:
Basic model of Gene Expression, Epigenetics
What is ChiP?
Why ChiP-qPCR?
Research Applications for ChIP PCR Arrays
Summary
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Sample & Assay Technologies
5. Basic Gene Expression Model
Protein “A”
Transcription
Initiation Complex
mRNA ”A”
Structural Gene “A”
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Sample & Assay Technologies
6. The Current Model
Activated
Transcription Factors
RNAi:
shRNA
siRNA
Protein “A”
NFκB
+
p53
Transcription
Initiation Complex
mRNA ”A”
–
Histones
p53 BS Me
Me
Me
Me Me
NFκB BS
DNA Methylation
Ac
Me
Histone-DNA
Interactions
Me Me
Structural Gene “A”
or Reporter system
DNA Methylation
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Sample & Assay Technologies
7. The Current Model
Activated
Transcription Factors
RNAi:
shRNA
miRNA
Protein “A”
NFκB
+
p53
Transcription
Initiation Complex
mRNA ”A”
–
Histones
p53 BS Me
Me
Protein-DNA
Interactions
Me
Me Me
NFκB BS
Ac
Me
Histone-DNA
Interactions
Me Me
Structural Gene “A”
TSS (+1) or Reporter system
DNA Methylation
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Sample & Assay Technologies
10. What IS ChIP-qPCR?
What is ChIP?
Chromatin Immuno-Precipitation
Method to examine in vivo protein-DNA interactions
• Transcription Factors, Modified Histones, Other nuclear factors
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What is qPCR?
qPCR = Quantitative Real-Time PCR
Faster, Higher Throughput than agarose gel electrophoresis
Gold-Standard Method to Detect DNA
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ChIP-qPCR is Chromatin Immunoprecipitation with qPCR Detection and Quantification
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Sample & Assay Technologies
11. What IS ChIP-qPCR?
What is ChIP?
Chromatin Immuno-Precipitation
Method to examine in vivo protein-DNA interactions
• Transcription Factors, Modified Histones, Other nuclear factors
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What is qPCR?
qPCR = Quantitative Real-Time PCR
Faster, Higher Throughput than agarose gel electrophoresis
Gold-Standard Method to Detect DNA
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ChIP-qPCR is Chromatin Immunoprecipitation with qPCR Detection and Quantification
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Sample & Assay Technologies
15. EpiTect qPCR Primers versus end-point PCR
Cycles
22
26
40
32
Semi-Quantitative PCR
IgG
Ab
Input
IgG
Ab
Input
IgG
Ab
Input
IgG
A
Ab
Input
ChIP-qPCR Primers:
• EVERY Site on EVERY Human,
Mouse, or Rat Promoter
• Specially Designed for Genomic
DNA Enriched from ChIP
• Validated Primer Specificity &
High Amplification Efficiency
B
Ct(dRn)
• Similar Amplification Efficiencies
for Each Primer Pair
Input
Ab
IgG
Assay Design:
• Based on 30 kb promoter tiling
• -20 kb to +10 kb relative to the
TSS
Cycles
Quantitative Real-Time PCR
• Each primer set targets 1 kb
region
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Sample & Assay Technologies
17. Applications for ChIP
Where does my Transcription Factor (or any protein that binds DNA) interact with
gDNA?
Example: Which p53 binding site is being used?
What is the chromatin structure (histone modifications)?
Example: Can p53 bind to this site?
Which question you are asking determines the experimental setup:
How many samples? (2 samples or 100?)
Which Antibody?
(is it validated for ChiP?)
Which method for detection? (microarray, sequencing, PCR, qPCR?)
Data Analysis?
Both questions may be necessary to fully understand gene regulation.
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Sample & Assay Technologies
18. Challenges Facing Biological Researchers
ChIP Procedure Related
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• Time-consuming (multiple days)
• Requires optimization of multiple steps
• Low DNA yields
Antibody Related
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• Screening for ChIP-grade suitability tedious & frustrating
DNA Quantification & Analysis Related
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•
•
•
•
Low throughput
Requires genomic DNA real-time PCR primer design experience
Requires positive & negative control assays
Tedious data analysis
Reproducible Results are a function of the Biological System and Technique
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Sample & Assay Technologies
19. EpiTect System – A Complete Solution
Removing Technique so Researchers can focus on the Biology
?
EpiTect One-Day Kit
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One-Day Protocol, Higher DNA Yield and Quality
Cross-Link
EpiTect-Grade Antibody Kits
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Validated ChIP-Grade Antibodies + Positive & Negative Controls
Sonication
EpiTect qPCR Arrays
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Multiple Assays & Controls Arranged in ONE PCR Plate
96 or 384 well Format Compatible with Most qPCR Instruments
Gene Regulation by Histone Modification
Custom Options Available
ChIP
EpiTect-qPCR Primers
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Primers for Every Site on Every Human, Mouse, or Rat Promoter
Validated & Wet-Bench Tested
Optimized SYBR Green Master Mix
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Data Analysis Software (Excel template)
qPCR
Data
Analysis
Answer
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Sample & Assay Technologies
20. Research Application Example I:
What is the mechanism of CDKN1A expression after 5-FU treatment?
5-FU treatment increases the expression of CDKN1A
Activates p53 transcription Factor
CDKN1A has p53 biniding sites (4 consensus sites!)
Research Questions:
Does p53 bind to the CDKN1A promoter?
Which of the 4 sites are used?
Are all 4 sites equally accessible? (chromatin strucutre questions)
How can we experimentally accomplish this?
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Sample & Assay Technologies
24. EpiTect Antibody Kits
For TF and Positive Controls
ChIP-Grade TF Antibody Kits
RNA Pol II
p53
Benefits:
• Experimentally Validated, Ready-to-Use
ChIP-Grade Antibodies & Control IgG
• High Efficiency and Low Background
• Includes Appropriate Positive & Negative
Control Real-Time qPCR Primers
The EpiTect ChIP qPCR System is compatible with any “ChIP grade” antibody
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Sample & Assay Technologies
26. EpiTect qPCR Assay Design: Tiling Scheme
TSS (+1)
Structural Gene
mRNA
gDNA
Distal Promoter
1 kb
Proximal Promoter
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1 kb
1 kb
qPCR Assay & Amplicon
1 kb
1 kb
Amplicon Coverage Range (Tile)
Thirty (30) 1-kb tiles from -20 kb to +10 kb relative to each TSS
Accommodate every human, mouse and rat gene promoter
Balance Between Sensitivity & Resolution
Quality Controlled for Specificity & Amplification Efficiency
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Sample & Assay Technologies
27. Wet Bench QC Validation of ALL PCR Arrays
All PCR Arrays are Wet-Bench 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: single melt-peak during dissociation
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Sample & Assay Technologies
28. Finding Your ChIP-qPCR Assays
EVERY Binding Site on EVERY Human, Mouse, Rat Promoter
Search by Gene
• Find primers for known or predicted nuclear factor binding sites relative to TSS
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Search by Transcription Factor and/or Gene
• Find primers for binding sites on transcription factor-targeted genes or predicted
binding sites in genes of interest
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Search by Chromosomal Position
• Find primers for specific locations from ChIP-on-chip or ChIP-Seq
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http://www.sabiosciences.com/chipqpcrsearch.php
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Sample & Assay Technologies
29. How the ChIP PCR Array System Works: Summary
• ChIP Fraction Preparation
• One-Day Kit & Antibody Kits
• 6 hours
• Load Plates (Preferably with
multi-channel or repeating
pipettors)
• 2 minutes
• Run 40 cycle qPCR Program
• 2 hours
• Upload and Analyze Data
• 15 minutes
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Sample & Assay Technologies
31. Experimental Treatments Alter Transcription
Factor Binding
Treatment with 5-FU increases p53 Binding to CDKN1A Promoter by 5- to 9-fold.
It also increases RNA Pol II binding to CDKN1A Promoter by 7.5-fold.
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Sample & Assay Technologies
32. Data Analysis
Data Analysis Principles:
Normalize ChIP Fraction Ct to Input DNA Fraction Ct Values
Percent of Input (or fraction enrichment) = 100 * 2 ^ -∆Ct
∆
Fold-Changes in DNA Binding = 2 ^ -∆∆ t
∆∆C
∆∆
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Free Data Analysis Excel Template:
http://www.sabiosciences.com/manuals/chipqpcranalysis.xls
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Sample & Assay Technologies
33. Are all of the p53 sites equally accessible?
P53 can interact with the -12, -3, -2 tiles but not -4
Is the chromatin structure different?
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Sample & Assay Technologies
35. EpiTect Antibody Kits
For Histone Modifications
ChIP-Grade Histone Antibody Kits
Antibody Name
Modifications
H3ac
H3K9ac
Acetylation
Benefits:
H4ac
H3K4me1
• Experimentally Validated, Ready-to-Use
ChIP-Grade Antibodies & Control IgG
H3K4me2
H3K4me3
• High Efficiency and Low Background
H3K9me1
H3K9me2
H3K9me3
Methylation
• Includes Appropriate Positive & Negative
Control Real-Time qPCR Primers
H3K27me3
H3K36me3
H3K79me3
H4K20me3
H3
Unmodified Control
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Sample & Assay Technologies
36. How the ChIP PCR Array System Works: Summary
• ChIP Fraction Preparation
• One-Day Kit & Antibody Kits
• 6 hours
• Load Plates (Preferably with
multi-channel or repeating
pipettors)
• 2 minutes
• Run 40 cycle qPCR Program
• 2 hours
• Upload and Analyze Data
• 15 minutes
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Sample & Assay Technologies
37. Modified Histone Distribution Across a Promoter
-1
Histone markers for actively transcribed genes (H3Ac and H3K4me2) but not for
transcriptionally inactive genes (H3K27me3) surround the CDKN1A gene making it
available to p53 regulation.
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Sample & Assay Technologies
38. Application Example 2: Chromatin structure for a pathway
Are changes in gene expression caused by Epigenetic Changes?
DNA Methylation
Chromatin Structure
miRNA
Methyl-Profiler
PCR Array
Epigenetic ChIP
PCR Array
miRNA
PCR Array
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Sample & Assay Technologies
40. How the ChIP PCR Array System Works: Summary
• ChIP Fraction Preparation
• One-Day Kit & Antibody Kits
• 6 hours
• Load Plates (Preferably with
multi-channel or repeating
pipettors)
• 2 minutes
• Run 40 cycle qPCR Program
• 2 hours
• Upload and Analyze Data
• 15 minutes
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Sample & Assay Technologies
41. EpiTect PCR Arrays
for Histone Regulation of Gene Expression
Analyze Pathway-Focused in vivo Protein-DNA Interactions
Using ChIP-enriched genomic DNA, simultaneously analyze DNAprotein interactions across a focused panel of genes representing an
epigenetically controlled biological pathway or disease state
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Evaluate association of histones or modified histones interacting with a
genomic region known for active transcriptional regulation
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Each array is a 96 or 384-well PCR plate containing experimentally
validated PCR primers (84 pathway-focused genes, 12 controls) that
target the proximal promoter region (+1 kb from the TSS) of each
gene
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Custom EpiTect qPCR Arrays Available
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Sample & Assay Technologies
43. Modified Histone Distribution Across a Promoter
Indication of OPEN chromatin and Transcription
-1
Histone markers for actively transcribed genes (H3Ac and H3K4me2) but not for
transcriptionally inactive genes (H3K27me3) surround the TSS
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Sample & Assay Technologies
44. EpiTect ChIP qPCR Array Layout
1
2
3
4
5
6
7
8
A
CDX2
DACH1
DLX1
DLX2
DNMT3B
EGR#
ESR1
EZH2
B
FOXP3
GATA1
GATA6
GLI2
C
HOXB13
HOXB3
HOXB5
D
HOXD4
HTR7
IRX4
E
NEUROD1
NFATC1
NKX2-2 NOTCH2
F
PITX3
POU4F1
POUF2
G
SP1
STAT1
STAT3
H
10
12
FOXA1 FOXA2 FOXP1 FOXP2
HOXC4
HOXC5 HOXC6 HOXC9 HOXD1 HOXD10
JUN
KLF2
KLF4
NRF2
OLIG2
PAX1
PAX5
POU5F1
PPARG
RB1
RUX1
SIX2
TBX5
TDGF1
TERT
TLX3
VDR
WRN
MYOD1 SERPINA1 SAT2
SAT
GAPDH(-3) GAPDH (+1) GAPDH+4 ALDOA
11
HAND1 HOXA10 HOXA11 HOXA2 HOXA3 HOXA7 HOXA9 HOXB1
HOXB8 HOXC10 HOXC12
ISL1
9
RPL30
Transcriptionally Active Genes
Transcriptionally Inactive Genes
Silenced Heterochromatin
Positive PCR Control
LIN28B LMX1B MSX2
MYC
NANOG
PAX9
PCNA
PITX2
SMAD2 SOX2
SOX6
SOX9
WT1
ZFPM2
ZIC1
IGX1A
PPC
PPC
PAX6
GAH-501
Human Stem Cell
Transcription Factors
Control primer sets include:
• System control to assess the qPCR performance (machine & handling).
• Different chromatin regions representing active and inactive chromatin regions to
evaluate the reliability of genomic DNA samples enriched by ChIP.
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Sample & Assay Technologies
45. EpiTect ChIP qPCR Array Layout
1
3
4
5
6
7
8
A
CDX2
DACH1
DLX1
DLX2
DNMT3B
EGR#
ESR1
EZH2
B
FOXP3
GATA1
GATA6
GLI2
C
HOXB13
HOXB3
HOXB5
D
HOXD4
HTR7
IRX4
E
NEUROD1
NFATC1
NKX2-2 NOTCH2
F
PITX3
POU4F1
POUF2
G
+1
Tile
ONLY
2
SP1
STAT1
STAT3
H
10
12
FOXA1 FOXA2 FOXP1 FOXP2
HOXC4
HOXC5 HOXC6 HOXC9 HOXD1 HOXD10
JUN
KLF2
KLF4
NRF2
OLIG2
PAX1
PAX5
POU5F1
PPARG
RB1
RUX1
SIX2
TBX5
TDGF1
TERT
TLX3
VDR
WRN
MYOD1 SERPINA1 SAT2
SAT
GAPDH(-3) GAPDH (+1) GAPDH+4 ALDOA
11
HAND1 HOXA10 HOXA11 HOXA2 HOXA3 HOXA7 HOXA9 HOXB1
HOXB8 HOXC10 HOXC12
ISL1
9
RPL30
Transcriptionally Active Genes
Transcriptionally Inactive Genes
Silenced Heterochromatin
Positive PCR Control
LIN28B LMX1B MSX2
MYC
NANOG
PAX9
PCNA
PITX2
SMAD2 SOX2
SOX6
SOX9
WT1
ZFPM2
ZIC1
IGX1A
PPC
PPC
PAX6
GAH-501
Human Stem Cell
Transcription Factors
Control primer sets include:
• System control to assess the qPCR performance (machine & handling).
• Different chromatin regions representing active and inactive chromatin regions to
evaluate the reliability of genomic DNA samples enriched by ChIP.
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Sample & Assay Technologies
46. EpiTect ChIP qPCR Arrays for Histone Regulation
of Gene Expression
Human and Mouse Cataloged Arrays Available:
Stem Cell Transcription Factors
Oncogene & Tumor Suppressor Genes
T Helper Cell Differentiation
Inflammatory Responses
Cancer Drug Targets
DNA Repair
Cell Cycle
Cell Lineage Identification
Polycomb & Trithorax Genes
Polycomb & Trithorax Complexes
Homeobox (HOX) Genes
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Custom EpiTect PCR Arrays
Promoter Binding Arrays – examine the promoter region of a particular gene
Gene Regulation Arrays – Follow up to ChIP-on-chip or ChIP-Seq
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Sample & Assay Technologies
47. EpiTect ChIP qPCR Arrays for Histone Regulation
of Gene Expression
Human and Mouse Cataloged Arrays Available:
Stem Cell Transcription Factors
Oncogene & Tumor Suppressor Genes
T Helper Cell Differentiation
Inflammatory Responses
Cancer Drug Targets
DNA Repair
Cell Cycle
Cell Lineage Identification
Polycomb & Trithorax Genes
Polycomb & Trithorax Complexes
Homeobox (HOX) Genes
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Custom EpiTect PCR Arrays
Promoter Binding Arrays – examine the promoter region of a particular gene
Gene Regulation Arrays – Follow up to ChIP-on-chip or ChIP-Seq
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Sample & Assay Technologies
49. Stem Cell Research Application Detail
____
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____
____
0 48048048 048048
_________ _________
Day
0
4
8 0
4
________
8 0 4
________
8 0 4
8
________
0
4 8
Nanog
Oct4
Sox2
Dlx1
Zfpm2
Neurod1
Hoxa3
Heat map visualization of the dynamic change
in pluripotency-associated gene expression
and histone modification during differentiation.
•H3Ac and H3K4me3 – transcriptionally active euchromatin
•H3K27me – transcriptionally inactive euchromatin
•H3K29me3 – silenced gene, densely packed heterochromatin
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Sample & Assay Technologies
50. Summary of the EpiTect System
?
EpiTect One-Day Kit
̣
One-Day Protocol, Higher DNA Yield and Quality
Cross-Link
EpiTect-Grade Antibody Kits
̣
Validated ChIP-Grade Antibodies + Positive & Negative Controls
Sonication
EpiTect PCR Arrays
̣
Multiple Assays & Controls Arranged in ONE PCR Plate
96 or 384 well Format Compatible with Most qPCR Instruments
Gene Regulation by Histone Modification
Custom Options Available
ChIP
EpiTect-qPCR Primers
̣
Primers for Every Site on Every Human, Mouse, or Rat Promoter
Validated & Wet-Bench Tested
Optimized SYBR Green Master Mix
̣
̣
Data Analysis Software (Excel template)
qPCR
Data
Analysis
Answer
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Sample & Assay Technologies
52. EpiTect ChIP qPCR Arrays for Histone Regulation
of Gene Expression
Human and Mouse Cataloged Arrays Available: (+1 TSS qPCR Assay for many Genes)
Apoptosis
Cell Cycle
DNA Repair
Homeobox (HOX) Genes
Polycomb & Trithorax Genes
Polycomb & Trithorax Complexes
T Helper Cell Differentiation
Cancer Drug Targets
Cell Lineage Identification
EMT (Epithelial to Mesenchymal Transition)
Inflammatory Responses
Oncogene & Tumor Suppressor Genes
Stem Cell Transcription Factors
Toll-like Receptor Signaling
Custom EpiTect ChIP qPCR Arrays
Promoter Binding Arrays – examine the promoter region of a particular gene
Gene Regulation Arrays – Follow up to ChIP-on-chip or ChIP-Seq
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Sample & Assay Technologies
53. EpiTect One-Day Kit Benefits
Speed & Ease
̣
Cross-Link
• One-day protocol
• Minimal steps to optimize
Universality
Sonication
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• Compatible with ANY ChIP-Grade antibody
Reliability & Consistency
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• Highly reproducible in different researchers’ hands
• qPCR quality DNA
ChIP
qPCR
Data
Analysis
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Sample & Assay Technologies
54. Solutions Provided by EpiTect qPCR System
Pathway, Disease or single loci Analysis using qPCR
• Genome wide qPCR Assays around every TSS in Human, Mouse
Rat genome
• Custom PCR Arrays can be manufactured to simultaneously
analyze up to 96 or 384 genomic loci
• Complement & follow-up for ChIP-on-chip & ChIP-Seq
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Reliable & Sensitive
• Stringent real-time PCR primer QC procedure
• As little as one million cells per ChIP assay (25-50 µg chromatin)
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Easy to Use:
• Simplified ChIP preparation protocol
• Accessible to any lab with access to qPCR instrument
• Simplified Data Analysis templates
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Sample & Assay Technologies
55. ChampionChIP qPCR System™
A Complete System from Chromatin Precipitation to Data Analysis
Outside US? Email SABio@qiagen.com for discount offers in your region.
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Sample & Assay Technologies
56. SciGlobe: Search a for your papers using a bigger
database than Pubmed and more relevant than Google Scholar
SciGlobe Advantage
•Free
•Weekly emails on
new papers
•Download and store
papers
•Export to citation
databases
•Larger database than
pubmed
•More relevant
searches than Google
scholar
Try it Now: http://www.sabiosciences.com/sciglobe.php
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Sample & Assay Technologies
57. EpiTect qPCR System™
A Complete System from Chromatin Precipitation to Data Analysis
Questions, Comments, Concerns?
US Applications Support
Questions, Comments, Concerns?
Global Applications Support
Discount offers? Register:
SABio@qiagen.com
888-503-3187
̣
support@sabiosciences.com
- 57 -
Sample & Assay Technologies