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Epi tect chi pqpcr_2013

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  • 1. EpiTect ChIP qPCR System A Complete System from Chromatin ImmunoPrecipitation to Data Analysis Samuel Rulli, Ph.D. Applications Scientist Samuel.Rulli@QIAGEN.com ̣ ̣ ̣ -1- 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 * -2- Sample & Assay Technologies
  • 3. Webinar Overview Introduction: Research Applications for ChIP PCR Arrays Summary -3- 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 -4- Sample & Assay Technologies
  • 5. Basic Gene Expression Model Protein “A” Transcription Initiation Complex mRNA ”A” Structural Gene “A” -5- 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 -6- 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 * -7- Sample & Assay Technologies
  • 8. Epigenetic Mechanisms of Gene Regulation Matouk, C. C. et al. Circ Res 2008;102:873-887 Copyright ©2008 American Heart Association -8- Sample & Assay Technologies
  • 9. Epigenetic Mechanisms of Gene Regulation TF Matouk, C. C. et al. Circ Res 2008;102:873-887 Copyright ©2008 American Heart Association -9- 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 ̣ ̣ 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 * - 10 - 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 ̣ ̣ 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 * - 11 - Sample & Assay Technologies
  • 12. Chromatin Immunoprecipitation Workflow - 12 - Sample & Assay Technologies
  • 13. Chromatin Immunoprecipitation Workflow Detection Methods/ Identification of DNA - 13 - Sample & Assay Technologies
  • 14. Chromatin Immunoprecipitation Workflow A Ct(dRn) B Input Ab IgG ChIP-qPCR Cycles - 14 - 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 - 15 - Sample & Assay Technologies
  • 16. Webinar Overview Introduction: Research Applications for ChIP PCR Arrays Regulation of CDKN1A Regulation of Stem Cell Differentiation Summary - 16 - 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. - 17 - Sample & Assay Technologies
  • 18. 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 - 18 - Sample & Assay Technologies
  • 19. 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 - 19 - 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? - 20 - Sample & Assay Technologies
  • 21. Chromatin Immunoprecipitation Workflow Stimulus X-Link Lyse Fragment X IP X X Reverse X-Link Purify DNA Input Anti-TF NIS Real-Time PCR Primers ChIP-qPCR - 21 - Sample & Assay Technologies
  • 22. EpiTect qPCR System: One-Day Kit - 22 - Sample & Assay Technologies
  • 23. EpiTect qPCR System: One-Day Kit - 23 - 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 - 24 - Sample & Assay Technologies
  • 25. EpiTect System: One-Day Kit - 25 - Sample & Assay Technologies
  • 26. 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 ̣ ̣ - 26 - 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 - 27 - 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 ̣ 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 - 28 - 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 - 29 - Sample & Assay Technologies
  • 30. Chromatin Immunoprecipitation Workflow 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 - 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. - 31 - 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 ∆∆ ̣ ̣ ̣ Free Data Analysis Excel Template: http://www.sabiosciences.com/manuals/chipqpcranalysis.xls - 32 - 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? - 33 - Sample & Assay Technologies
  • 34. Chromatin Immunoprecipitation Workflow 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 - 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 - 35 - 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 - 36 - 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. - 37 - 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 - 38 - Sample & Assay Technologies
  • 39. 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 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 - 40 - 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 ̣ 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 ̣ ̣ Custom EpiTect qPCR Arrays Available - 41 - Sample & Assay Technologies
  • 42. Modified Histone Distribution Across a Promoter -1 - 42 - 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 - 43 - 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. - 44 - 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. - 45 - 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 ̣ 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 - 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 ̣ 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 ̣ - 47 - Sample & Assay Technologies
  • 48. 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 Sample & Assay Technologies
  • 49. 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 - 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 - 50 - Sample & Assay Technologies
  • 51. Webinar Overview Introduction Research Applications for ChIP PCR Arrays Summary - 51 - 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 ̣ ̣ - 52 - Sample & Assay Technologies
  • 53. 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 - 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 ̣ 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 - 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. - 55 - 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 - 56 - 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

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