Epi tect chi pqpcr_2013

Sample & Assay Technologies- 1 -

EpiTect ChIP qPCR System

A Complete System from Chromatin ImmunoPrecipitation to Data Analysis

Samuel Rulli, Ph.D.Applications [email protected]


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A Complete System from Chromatin ImmunoPrecipitation to Data Analysis

EpiTect ChIP qPCR System

Questions, Comments, Concerns?US Applications Support

[email protected]

Questions, Comments, Concerns?Global Applications Support

[email protected]


Webinar Overview

� Introduction:

� Research Applications for ChIP PCR Arrays

� Summary


Webinar Overview

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


� Summary


Basic Gene Expression Model

Structural Gene “A”

mRNA ”A”

Protein “A”

Transcription Initiation Complex


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:shRNAsiRNA


+

–


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

+

–

Activated Transcription Factors


p53 BS NFκB BS

p53

NFκB

Histones

Histone-DNA Interactions

Me AcMeMe

Protein “A”

mRNA ”A”

DNA Methylation

Me MeMeMe Me

RNAi:shRNAmiRNA

The Current Model

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Protein-DNA Interactions


Epigenetic Mechanisms of Gene Regulation

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


Epigenetic Mechanisms of Gene Regulation

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

TF


What IS ChIP-qPCR?

What is ChIP?Chromatin Immuno- PrecipitationMethod to examine in vivo protein-DNA interactions

• Transcription Factors, Modified Histones, Other nuclear factors

What is qPCR?qPCR = Quantitative Real-Time PCRFaster, Higher Throughput than agarose gel electrop horesisGold-Standard Method to Detect DNA

ChIP-qPCR is Chromatin Immunoprecipitation with qPCR Detection and Quantification

*


What IS ChIP-qPCR?

What is ChIP?Chromatin Immuno- PrecipitationMethod to examine in vivo protein-DNA interactions

• Transcription Factors, Modified Histones, Other nuclear factors

What is qPCR?qPCR = Quantitative Real-Time PCRFaster, Higher Throughput than agarose gel electrop horesisGold-Standard Method to Detect DNA

ChIP-qPCR is Chromatin Immunoprecipitation with qPCR Detection and Quantification

*


Chromatin Immunoprecipitation Workflow



Detection Methods/ Identification of DNA


Cycles

Ct(

dRn)

Input Ab IgG

A

B

ChIP-qPCR



EpiTect qPCR Primers versus end-point PCR

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

• Similar Amplification Efficiencies for Each Primer Pair

Cycles

Ct(

dRn) Input Ab IgG

B

Quantitative Real-Time PCRCycles

Ct(

dRn) Input Ab IgG

Cycles 22

IgG

Ab

Inpu

t

A

26 32 40Ig

G

Ab

Inpu

t

IgG

Ab

Inpu

t

IgG

Ab

Inpu

t

Semi-Quantitative PCR

Assay Design:

• Based on 30 kb promoter tiling

• -20 kb to +10 kb relative to the TSS

• Each primer set targets 1 kb region


Webinar Overview

� Introduction:

� Research Applications for ChIP PCR Arrays� Regulation of CDKN1A � Regulation of Stem Cell Differentiation

� Summary


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.


Challenges Facing Biological Researchers

ChIP Procedure Related• Time-consuming (multiple days)• Requires optimization of multiple steps• Low DNA yields

Antibody Related• Screening for ChIP-grade suitability tedious & frustrating

DNA Quantification & Analysis Related• 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


EpiTect System – A Complete Solution

EpiTect One-Day Kit� One-Day Protocol, Higher DNA Yield and Quality

EpiTect-Grade Antibody Kits� Validated ChIP-Grade Antibodies + Positive & Negative Controls

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

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)

Cross-Link

Sonication

ChIP

qPCR

DataAnalysis

Removing Technique so Researchers can focus on the Biology

?

Answer


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?



XX

X

X-LinkLyseFragment IP

Reverse X-LinkPurify DNA

Stimulus

Input Anti-TF NIS

ChIP-qPCR

Real-TimePCR Primers


EpiTect qPCR System: One-Day Kit


EpiTect qPCR System: One-Day Kit


EpiTect Antibody KitsFor 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


EpiTect System: One-Day Kit


EpiTect qPCR Assay Design: Tiling Scheme

Thirty (30) 1-kb tiles from -20 kb to +10 kb relative to each TSSAccommodate every human, mouse and rat gene promoterBalance Between Sensitivity & ResolutionQuality Controlled for Specificity & Amplification Efficiency

qPCR Assay & Amplicon Amplicon Coverage Range (Tile)

Proximal Promoter

TSS (+1)mRNA

gDNA

Structural Gene

Distal Promoter

1 kb 1 kb1 kb 1 kb1 kb


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


Finding Your ChIP -qPCR Assays

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

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

EVERY Binding Site on EVERY Human, Mouse, Rat Promo ter


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



XX

X



Stimulus

Input Anti-TF NIS

ChIP-qPCR


TF-specific and non-specific IP are done in paralle l


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.


Data Analysis

Data Analysis Principles:Normalize ChIP Fraction C t to Input DNA Fraction C t Values Percent of Input (or fraction enrichment) = 100 * 2 ^ -∆∆∆∆Ct

Fold-Changes in DNA Binding = 2 ^ - ∆∆∆∆∆∆∆∆Ct

Free Data Analysis Excel Template:

http://www.sabiosciences.com/manuals/chipqpcranalys is.xls


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?



XX

X



Stimulus

Input Anti-TF NIS

ChIP-qPCR



EpiTect Antibody KitsFor Histone Modifications

ChIP-Grade Histone Antibody Kits

Antibody Name Modifications

H3ac

AcetylationH3K9ac

H4ac

H3K4me1

Methylation

H3K4me2

H3K4me3

H3K9me1

H3K9me2

H3K9me3

H3K27me3

H3K36me3

H3K79me3

H4K20me3

H3 Unmodified Control

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








Modified Histone Distribution Across a Promoter

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.

-1


Application Example 2: Chromatin structure for a pa thway

Are changes in gene expression caused by Epigenetic Changes?

DNA Methylation Chromatin Structure miRNA

Methyl-ProfilerPCR Array

Epigenetic ChIPPCR Array

miRNAPCR Array


Stem Cell Research

P19 Cells

Day 1 4 8

mRNA H3ac H3K4me3 H3K27me3 H3K9me3Pou5f1/Oct4

1 4 81 4 81 4 81 4 8

Active Markers Inactive or Repressing Markers








EpiTect PCR Arraysfor Histone Regulation of Gene Expression

Using ChIP-enriched genomic DNA, simultaneously analyze DNA-protein interactions across a focused panel of genes representing an epigenetically controlled biological pathway or dis ease state

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

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

Custom EpiTect qPCR Arrays Available

Analyze Pathway-Focused in vivo Protein-DNA Interactions



-1



Histone markers for actively transcribed genes (H3Ac and H3K4me2) but not for transcriptionally inactive genes (H3K27me3) surround the TSS

-1

Indication of OPEN chromatin and Transcription


EpiTect ChIP qPCR Array Layout

GAH-501

Human Stem Cell Transcription Factors

1 2 3 4 5 6 7 8 9 10 11 12

A CDX2 DACH1 DLX1 DLX2 DNMT3B EGR# ESR1 EZH2 FOXA1 FOXA2 FOXP1 FOXP2

B FOXP3 GATA1 GATA6 GLI2 HAND1 HOXA10 HOXA11 HOXA2 HOXA3 HOXA7 HOXA9 HOXB1

C HOXB13 HOXB3 HOXB5 HOXB8 HOXC10 HOXC12 HOXC4 HOXC5 HOXC6 HOXC9 HOXD1 HOXD10

D HOXD4 HTR7 IRX4 ISL1 JUN KLF2 KLF4 LIN28B LMX1B MSX2 MYC NANOG

E NEUROD1 NFATC1 NKX2-2 NOTCH2 NRF2 OLIG2 PAX1 PAX5 PAX6 PAX9 PCNA PITX2

F PITX3 POU4F1 POUF2 POU5F1 PPARG RB1 RUX1 SIX2 SMAD2 SOX2 SOX6 SOX9

G SP1 STAT1 STAT3 TBX5 TDGF1 TERT TLX3 VDR WRN WT1 ZFPM2 ZIC1

H GAPDH(-3) GAPDH (+1) GAPDH+4 ALDOA RPL30 MYOD1 SERPINA1 SAT2 SAT IGX1A PPC PPC

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.

Transcriptionally Active Genes

Transcriptionally Inactive Genes

Silenced Heterochromatin

Positive PCR Control


GAH-501

Human Stem Cell Transcription Factors

1 2 3 4 5 6 7 8 9 10 11 12

A CDX2 DACH1 DLX1 DLX2 DNMT3B EGR# ESR1 EZH2 FOXA1 FOXA2 FOXP1 FOXP2

B FOXP3 GATA1 GATA6 GLI2 HAND1 HOXA10 HOXA11 HOXA2 HOXA3 HOXA7 HOXA9 HOXB1

C HOXB13 HOXB3 HOXB5 HOXB8 HOXC10 HOXC12 HOXC4 HOXC5 HOXC6 HOXC9 HOXD1 HOXD10

D HOXD4 HTR7 IRX4 ISL1 JUN KLF2 KLF4 LIN28B LMX1B MSX2 MYC NANOG

E NEUROD1 NFATC1 NKX2-2 NOTCH2 NRF2 OLIG2 PAX1 PAX5 PAX6 PAX9 PCNA PITX2

F PITX3 POU4F1 POUF2 POU5F1 PPARG RB1 RUX1 SIX2 SMAD2 SOX2 SOX6 SOX9

G SP1 STAT1 STAT3 TBX5 TDGF1 TERT TLX3 VDR WRN WT1 ZFPM2 ZIC1

H GAPDH(-3) GAPDH (+1) GAPDH+4 ALDOA RPL30 MYOD1 SERPINA1 SAT2 SAT IGX1A PPC PPC

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.

Transcriptionally Active Genes

Transcriptionally Inactive Genes

Silenced Heterochromatin

Positive PCR Control

+1 Tile ONLY

EpiTect ChIP qPCR Array Layout


EpiTect ChIP qPCR Arrays for Histone Regulationof 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



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


Stem Cell Research

P19 Cells

Day 1 4 8

mRNA H3ac H3K4me3 H3K27me3 H3K9me3Pou5f1/Oct4

1 4 81 4 81 4 81 4 8

Active Markers Inactive or Repressing Markers


0 4 8 0 4 8 0 4 8 0 4 8 0 4 8

Nanog

Dlx1Zfpm2

Neurod1Hoxa3

Sox2Oct4

Heat map visualization of the dynamic change in pluripotency-associated gene expression

and histone modification during differentiation.

•H3Ac and H3K4me3 – transcriptionally active euchroma tin

•H3K27me – transcriptionally inactive euchromatin

•H3K29me3 – silenced gene, densely packed heterochrom atin

Day 0 4 8 0 4 8 0 4 8 0 4 8 0 4 8

_________ _________ ________ ________ ________

____ ____ ____ ____ ____

Stem Cell Research Application Detail


Summary of the EpiTect System

EpiTect One-Day Kit� One-Day Protocol, Higher DNA Yield and Quality

EpiTect-Grade Antibody Kits� Validated ChIP-Grade Antibodies + Positive & Negative Controls

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

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)

Cross-Link

Sonication

ChIP

qPCR

DataAnalysis

?

Answer


Webinar Overview

� Introduction


� Summary



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


EpiTect One-Day Kit Benefits

Speed & Ease• One-day protocol• Minimal steps to optimize

Universality• Compatible with ANY ChIP-Grade antibody

Reliability & Consistency• Highly reproducible in different researchers’ hands• qPCR quality DNA

Cross-Link

Sonication

ChIP

qPCR

DataAnalysis


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


ChampionChIP qPCR System™

A Complete System from Chromatin Precipitation to D ata Analysis

Outside US? Email [email protected] for discount offers in your region.


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


EpiTect qPCR System™

A Complete System from Chromatin Precipitation to D ata Analysis

Questions, Comments, Concerns?US Applications Support

[email protected]

Questions, Comments, Concerns?Global Applications SupportDiscount offers? Register:

[email protected]

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