Epi tect chi pqpcr_2013

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

EpiTect ChIP qPCR System
A Complete System from Chromatin
ImmunoPrecipitation to Data Analysis

Questions, Comments, Concerns?
US Applications Support

Global Applications Support
SABio@qiagen.com

888-503-3187
̣

support@sabiosciences.com
*
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Webinar Overview

Introduction:
Research Applications for ChIP PCR Arrays
Summary

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Webinar Overview

Introduction:
Basic model of Gene Expression, Epigenetics
What is ChiP?
Why ChiP-qPCR?
Summary

-4-


Basic Gene Expression Model

Protein “A”

Transcription
Initiation Complex

mRNA ”A”

Structural Gene “A”

-5-


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

or Reporter system

DNA Methylation

-6-


The Current Model
Activated

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

TSS (+1) or Reporter system

DNA Methylation

*
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Epigenetic Mechanisms of Gene Regulation

Matouk, C. C. et al. Circ Res 2008;102:873-887
Copyright ©2008 American Heart Association

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Epigenetic Mechanisms of Gene Regulation

TF

Matouk, C. C. et al. Circ Res 2008;102:873-887
Copyright ©2008 American Heart Association

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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
̣

̣

What is qPCR?
qPCR = Quantitative Real-Time PCR
Faster, Higher Throughput than agarose gel electrophoresis
Gold-Standard Method to Detect DNA
̣

̣

̣

ChIP-qPCR is Chromatin Immunoprecipitation with qPCR Detection and Quantification
*
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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
̣

̣

What is qPCR?
qPCR = Quantitative Real-Time PCR
Faster, Higher Throughput than agarose gel electrophoresis
Gold-Standard Method to Detect DNA
̣

̣

̣

ChIP-qPCR is Chromatin Immunoprecipitation with qPCR Detection and Quantification
*
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Chromatin Immunoprecipitation Workflow

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Detection Methods/ Identification of DNA

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A

Ct(dRn)

B
Input

Ab

IgG

ChIP-qPCR

Cycles

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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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Webinar Overview

Introduction:
Regulation of CDKN1A
Regulation of Stem Cell Differentiation
Summary

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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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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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EpiTect System – A Complete Solution
Removing Technique so Researchers can focus on the Biology
?

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 qPCR 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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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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Stimulus
X-Link
Lyse
Fragment

X

IP

X

X
Reverse X-Link
Purify DNA
Input

Anti-TF

NIS
Real-Time
PCR Primers

ChIP-qPCR

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EpiTect qPCR System: One-Day Kit

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EpiTect qPCR System: One-Day Kit

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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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EpiTect System: One-Day Kit

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EpiTect qPCR Assay Design: Tiling Scheme
TSS (+1)

Structural Gene
mRNA
gDNA

Distal Promoter
1 kb

Proximal Promoter

̣

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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̣

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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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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
̣

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
̣

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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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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Stimulus
X-Link
Lyse
Fragment

X

IP

X

X
Reverse X-Link
Purify DNA
Input

Anti-TF

NIS
Real-Time
PCR Primers

ChIP-qPCR
TF-specific and non-specific IP are done in parallel

- 30 -


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.

- 31 -


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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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?

- 33 -


Stimulus
X-Link
Lyse
Fragment

X

IP

X

X
Reverse X-Link
Purify DNA
Input

Anti-TF

NIS
Real-Time
PCR Primers

ChIP-qPCR

- 34 -


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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• 6 hours
pipettors)
• 2 minutes
• 2 hours
• 15 minutes

- 36 -


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.
- 37 -


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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Stem Cell Research

P19 Cells
H3ac

mRNA

H3K4me3 H3K27me3 H3K9me3

Pou5f1/Oct4
Day 1

4

8 1

4

8

1

4

Active Markers

- 39 -

8 1

4

8

1

4

8

Inactive or Repressing Markers


• 6 hours
pipettors)
• 2 minutes
• 2 hours
• 15 minutes

- 40 -


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
̣

Evaluate association of histones or modified histones interacting with a
genomic region known for active transcriptional regulation
̣

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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-1

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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

- 43 -


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

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.

- 44 -


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

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.

- 45 -


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
̣

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
̣

- 46 -


of Gene Expression
Human and Mouse Cataloged Arrays Available:
Cancer Drug Targets
DNA Repair
Cell Cycle
̣

Custom EpiTect PCR Arrays
̣

- 47 -


Stem Cell Research

P19 Cells
H3ac

mRNA

H3K4me3 H3K27me3 H3K9me3

Pou5f1/Oct4
Day 1

4

8 1

4

8

1

4

Active Markers

- 48 -

8 1

4

8

1

4

8

Inactive or Repressing Markers


Stem Cell Research Application Detail

____

____ ____

____

____

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

- 49 -


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

- 50 -


Webinar Overview

Introduction
Summary

- 51 -


of Gene Expression
Human and Mouse Cataloged Arrays Available: (+1 TSS qPCR Assay for many Genes)
Apoptosis
Cell Cycle
DNA Repair
Cancer Drug Targets
EMT (Epithelial to Mesenchymal Transition)
Toll-like Receptor Signaling
Custom EpiTect ChIP qPCR Arrays
̣

̣

- 52 -


EpiTect One-Day Kit Benefits
Speed & Ease
̣

Cross-Link

• One-day protocol
• Minimal steps to optimize

Universality

Sonication

̣

• Compatible with ANY ChIP-Grade antibody

Reliability & Consistency
̣

• Highly reproducible in different researchers’ hands
• qPCR quality DNA

ChIP

qPCR
Data
Analysis

- 53 -


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
̣

Reliable & Sensitive
• Stringent real-time PCR primer QC procedure
• As little as one million cells per ChIP assay (25-50 µg chromatin)
̣

Easy to Use:
• Simplified ChIP preparation protocol
• Accessible to any lab with access to qPCR instrument
• Simplified Data Analysis templates
̣

- 54 -


ChampionChIP qPCR System™
A Complete System from Chromatin Precipitation to Data Analysis

Outside US? Email SABio@qiagen.com for discount offers in your region.
- 55 -


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

- 56 -


EpiTect qPCR System™
A Complete System from Chromatin Precipitation to Data Analysis

US Applications Support

Global Applications Support
Discount offers? Register:
SABio@qiagen.com

888-503-3187
̣

support@sabiosciences.com

- 57 -


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