Epigenetics 2013

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Epigenetics 2013

  1. 1. Understanding Gene Expression and Epigenetics Using a Pathway Approach: PCR Array Systems for Gene & MicroRNA Expression Profiling CpG Island Methylation Analysis and ChiP Samuel J. Rulli Jr., Ph.D. qPCR Applications Scientist Samuel.Rulli@QIAGEN.com The PCR Arrays are intended for molecular biology applications. This product is not Intended for the diagnosis, prevention, or treatment of disease. Profiling genes by pathways and diseases -1- Sample & Assay Technologies
  2. 2. Understanding Gene Expression and Epigenetics Using a Pathway Approach: PCR Array Systems for Gene & MicroRNA Expression Profiling CpG Island Methylation Analysis and ChiP Questions, Comments, Concerns? US Applications Support support@sabiosciences.com Questions, Comments, Concerns? Global Applications Support SABio@qiagen.com The PCR Arrays are intended for molecular biology applications. This product is not Intended for the diagnosis, prevention, or treatment of disease. Profiling genes by pathways and diseases -2- Sample & Assay Technologies
  3. 3. Seminar Overview Cancer Research Apoptosis What is Epigenetics? PCR Arrays for Gene Expression Profiling Tumor and Metastatic Tumor Cells Cell Cycle Inflammation ECM/Adhesion Neuroscience Signal Transduction PCR Arrays for Epigenetic Applications Profiling miRNA Biomarkers in Colon Cancer Profiling DNA Methylation in Breast Cancer Cell lines and Normal Breast Tissue Profiling Chromatin Structure through Histone Modifications Summary and Questions Stem Cell/ Development Toxicology/ Drug ADME The PCR Arrays are intended for molecular biology applications. This product is not Intended for the diagnosis, prevention, or treatment of disease. Profiling genes by pathways and diseases -3- Sample & Assay Technologies
  4. 4. Seminar Overview Cancer Research Apoptosis What is Epigenetics? PCR Arrays for Gene Expression Profiling Tumor and Metastatic Tumor Cells Cell Cycle Inflammation ECM/Adhesion Neuroscience Signal Transduction PCR Arrays for Epigenetic Applications Profiling miRNA Biomarkers in Colon Cancer Profiling DNA Methylation in Breast Cancer Cell lines and Normal Breast Tissue Profiling Chromatin Structure through Histone Modifications Summary and Questions Stem Cell/ Development Toxicology/ Drug ADME The applications presented here are for research purposes. Not for diagnostic use. Profiling genes by pathways and diseases -4- Sample & Assay Technologies
  5. 5. What is Epigenetics? Epigenetics: “Epi” from Greek meaning “above or over genetics” Biological Definition: Something that effects the Phenotype without changing the Genotype; Something that can effect gene expression without causing a mutation in the DNA code Profiling genes by pathways and diseases -5- Sample & Assay Technologies
  6. 6. Current View of Gene Expression Activated Transcription Factors RNAi: miRNA Protein “A” NFκB + p53 Transcription Initiation Complex mRNA ”A” – Histones Histones Structural Gene “A” Me p53 BS AC AC Me NFκB BS Me Me Me Me Me Me Me TSS (+1) Gene Expression is Complex with multiple layers of control Profiling genes by pathways and diseases -6- Sample & Assay Technologies
  7. 7. Multiple Epigenetic mechanisms influence Gene Expression Activated Transcription Factors RNAi: miRNA NFκB p53 Transcription Initiation Complex mRNA ”A” Histones Histones Structural Gene “A” Me p53 BS AC AC Me NFκB BS Me Me Me Me Me Me Me TSS (+1) Gene Expression is Complex with multiple layers of control Profiling genes by pathways and diseases -7- Sample & Assay Technologies
  8. 8. PCR Arrays for Gene Expression and Epigenetic Regulation Activated Transcription Factors RNAi: miRNA Protein “A” NFκB p53 Transcription Initiation Complex mRNA ”A” Histones Histones Structural Gene “A” Me p53 BS AC AC Me NFκB BS Me Me Me Me Me Me Me TSS (+1) Gene Expression is Complex with multiple layers of control Profiling genes by pathways and diseases -8- Sample & Assay Technologies
  9. 9. Epigenetics effects multiple pathways and diseases Biological Mechanisms of Epigenetics: miRNA Regulates translation or stability of mRNA DNA Methylation: Regulates transcription factor access, chromatin structure Histone modifications: Regulates bulk chromatin structure and transcription factor access Biological Pathways and Diseases Effected by Epigenetics: Cancer- methylation of tumor suppressor genes Differentiation- changes in the histone code Immunology: miRNA regulation of cell cycle proteins by viruses Almost all pathways and diseases may have an underlying Epigenetic component HOW CAN RESEARCHERS PROFILE GENE EXPRESSION AND EPIGENETIC MECHANISMS? Profiling genes by pathways and diseases -9- Sample & Assay Technologies
  10. 10. ANSWER: PCR Arrays WHAT IS A PCR ARRAY? Collection of SYBR-Green based qPCR Assays with biological content with Focus on a Disease or Pathway Available for any qPCR instrument Available for a variety of laboratory applications: mRNA profiling (Gene Expression) miRNA profiling DNA Methylation Chromatin Precipitation Part of a complete “System” Sample Isolation to Final Data Publishable Data Component Profiling genes by pathways and diseases - 10 - Sample & Assay Technologies
  11. 11. 5 Components in the PCR Array System PCR ARRAYS are a Complete System for Pathway-Focused Gene Expression Analysis 1. Sample Isolation (QIAGEN RNeasy)* 2. RT2 PCR Arrays (96, 384 and Rotorgene Q formats) 3. RT2 First Strand kit** 4. RT2 SYBR Green Master Mix 5. Free data analysis software (web or excel-based) Publishable story Ease of use Relevant genes Accurate Reliable Complete System from Sample Preparation to Fold Change Results *substitute QIAGEN miRNeasy ** substitute miScriptII RT Kit for miRNA *substitute QIAGEN DNeasy Blood and Tissue; **substitute EpiTect Methyl II RE Kit Profiling genes by pathways and diseases - 11 - Sample & Assay Technologies
  12. 12. Seminar Overview Cancer Research Apoptosis What is Epigenetics? PCR Arrays for Gene Expression Profiling Tumor and Metastatic Tumor Cells Cell Cycle Inflammation ECM/Adhesion Neuroscience Signal Transduction PCR Arrays for Epigenetic Applications Profiling miRNA Biomarkers in Colon Cancer Profiling DNA Methylation in Breast Cancer Cell lines and Normal Breast Tissue Profiling Chromatin Structure through Histone Modifications Summary and Questions Stem Cell/ Development Toxicology/ Drug ADME The applications presented here are for research purposes. Not for diagnostic use. Profiling genes by pathways and diseases - 12 - Sample & Assay Technologies
  13. 13. PCR Arrays for Gene Expression Cancer Research Research Question: What are the gene expression differences between a metastatic and non-metastatic Breast Tumor? Profiling genes by pathways and diseases - 13 - Sample & Assay Technologies
  14. 14. Anatomy of a RT2 Profiler PCR Array (for mRNA Profiling) 84 Pathway-Specific Genes of Interest . 5 Housekeeping Genes . Genomic DNA Contamination Control . Reverse Transcription Controls (RTC) . Positive PCR Controls (PPC) . Profiling genes by pathways and diseases - 14 - Sample & Assay Technologies
  15. 15. Anatomy of a RT2 Profiler PCR Array (for mRNA Profiling) 84 Pathway-Specific Genes of Interest . 5 Housekeeping Genes . Genomic DNA Contamination Control . Reverse Transcription Controls (RTC) . Positive PCR Controls (PPC) . . Profiling genes by pathways and diseases - 15 - Sample & Assay Technologies
  16. 16. Built-in Standard and Performance Controls 5 Housekeeping Genes Controls Flexibility - researchers select the most stable reference (“housekeeping”) gene for their experimental situation Human: GAPDH, ACTB, B2M (Beta-2-Microglobulin), HPRT1 (Hypoxanthine Phosphoribosyltransferase 1), RPL13A (Ribosomal Protein 13A) Sample and qPCR Performance Controls Reverse Transcription Controls (RTC) Control for impurities that may degrade RNA or inhibit RT enzyme Positive PCR Controls (PPC) Control for inhibitors of the PCR step Used in conjunction with RTC assay to establish RT efficiency Genomic DNA Contamination Control (GDC) Detect gDNA contamination – Single exon genes important for biology (can’t design cross-intron qPCR Assays) – Pseudo-genes confound analysis of low expressing genes (detected with cross-intron assays) – Incorporated DNA removal step in cDNA synthesis kit to remove residual genomic DNA Profiling genes by pathways and diseases - 16 - Sample & Assay Technologies
  17. 17. How RT2 Profiler PCR Arrays Work cDNA Synthesis (C-03 kit) – Genomic DNA Removal Step (5 min.) – Reverse Transcription Step (20 min.) . Load Plates 1 Sample per PCR Array 2 minutes with multi-channel pipet . Run 40 cycle qPCR Program Standard cycling conditions for all Real Time PCR Instruments 2 hours . Upload and Analyze Data 15 minutes from Raw Ct to Fold Change Data . Profiling genes by pathways and diseases - 17 - Sample & Assay Technologies
  18. 18. How RT2 Profiler PCR Arrays Work cDNA Synthesis (C-03 kit) – Genomic DNA Removal Step (5 min.) – Reverse Transcription Step (20 min.) . Load Plates 1 Sample per PCR Array 2 minutes with multi-channel pipet . Run 40 cycle qPCR Program Standard cycling conditions for all Real Time PCR Instruments 2 hours . Upload and Analyze Data 15 minutes from Raw Ct to Fold Change Data . Profiling genes by pathways and diseases - 18 - Sample & Assay Technologies
  19. 19. How RT2 Profiler PCR Arrays Work cDNA Synthesis (C-03 kit) – Genomic DNA Removal Step (5 min.) – Reverse Transcription Step (20 min.) . Load Plates 1 Sample per PCR Array 2 minutes with multi-channel pipet . Run 40 cycle qPCR Program Standard cycling conditions for all Real Time PCR Instruments 2 hours . Upload and Analyze Data 15 minutes from Raw Ct to Fold Change Data . Profiling genes by pathways and diseases - 19 - Sample & Assay Technologies
  20. 20. How RT2 Profiler PCR Arrays Work cDNA Synthesis (C-03 kit) – Genomic DNA Removal Step (5 min.) – Reverse Transcription Step (20 min.) . Load Plates 1 Sample per PCR Array 2 minutes with multi-channel pipet . Run 40 cycle qPCR Program Standard cycling conditions for all Real Time PCR Instruments 2 hours . Upload and Analyze Data 15 minutes from Raw Ct to Fold Change Data . Profiling genes by pathways and diseases - 20 - Sample & Assay Technologies
  21. 21. Clustergram Data Analysis Web-Based Software Scatter Plot No installation or licensing needed . From Raw Ct Values to Fold Change Results ∆∆C(t) Method . Multiple Analysis Formats Scatter Plot Volcano Plot Multi-Group Plot Clustergram . Volcano Plot Also available as downloadable Excel templates Profiling genes by pathways and diseases - 21 - Sample & Assay Technologies
  22. 22. Wet Bench QC Validation of ALL qPCR Assays on every PCR Array All PCR Arrays are Wet-Bench Validated qPCR Assays on 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 Validation performed on each lot (synthesized oligonucleotides) of Primers Profiling genes by pathways and diseases - 22 - Sample & Assay Technologies
  23. 23. PCR Arrays for All Biomedical Researchers ~ 100 different pathways for Human, Mouse, Rat, Monkey and Fly. Cancer and Apoptosis Cytokines & Inflammation Development & Stem Cells Apoptosis Inflammatory Cytokines Stem Cells Cell Cycle Th17 for Inflammation (NEW!) Wnt Signaling Human miRNA Array (NEW!) Common Cytokines Notch Signaling Breast Cancer & Estrogen Receptor Chemokines TGFβ / BMP Signaling Tumor Metastasis NF-κB Signaling Pathway Endothelial Cell Biology Cancer PathwayFinder Th1-Th2-Th3 Osteogenesis Angiogenesis TNF Ligands Growth Factors Cancer Drug Resistance Toll-like Receptors ECM & Adhesion Signal Transduction Toxicology & Drug Metabolism Neuroscience Signal Transduction PathwayFinder Drug Metabolism Neuroscience Ion Channels NFkB Signaling Drug Phase I Enzymes Neurotransmitter Receptors Jak / Stat Signaling Drug Transporters Neurotrophins & Receptors DNA Damage Signaling Oxidative Stress Neurogenesis and Neural Stem Cell Insulin Signaling Stress & Toxicity MAP Kinase Signaling Other Diseases Custom PCR Arrays cAMP / Calcium Signaling Atherosclerosis 96-Well Custom Arrays p53 Signaling Diabetes 384-Well Custom Arrays Complete PCR Array List Profiling genes by pathways and diseases - 23 - Sample & Assay Technologies
  24. 24. How Genes on PCR Arrays Are Selected Biologically relevant gene content . Not simply biochemical pathways or kinase cascades Published association with the biological or disease pathway Specific gene information is gathered from a large number of different overlapping sources including: - Multiple Publicly Accessible Databases - Text Mining Relevant Literature Technically relevant gene content Use genes that are regulated at the mRNA level Specific feedback from thought leaders . Genes with High Relevance Profiling genes by pathways and diseases - 24 - Sample & Assay Technologies
  25. 25. Customizable PCR Arrays Analyze the Genes Most Important To Your Research . . Available for any real-time PCR machine. Wet-Bench Validated design and performance. 2 to 3 week turn around time. Same Data Analysis as Catalogued Arrays Profiling genes by pathways and diseases - 25 - Sample & Assay Technologies
  26. 26. PCR Arrays for All Biomedical Researchers ~ 100 different pathways for Human, Mouse, Rat, Monkey and Fly. Cancer Research Research Question: What are the gene expression differences between a metastatic and non-metastatic Breast Tumor? Angiogenesis Epithelial to Mesenchymal Transition Tumor Metastasis Cancer Drug Resistance p53 Signaling Pathway Wnt Signaling Pathway Cell Cycle Breast Cancer and Estrogen Receptor Signaling DNA Damage Signaling Pathway MAP Kinase Signaling Pathway Angiogenic Growth Factors and Inhibitors Protein Phosphatases PI3K-AKT Signaling Pathway Profiling genes by pathways and diseases - 26 - Sample & Assay Technologies
  27. 27. Application Example Pathway Focused Analysis between metastatic and non-metastatic tumors What is the gene expression differences between a metastatic and non-metastatic Breast Tumor? Tumor Metastasis PCR Array: • • • • • • • • • • • • • Cell Adhesion Matrix Metalloproteinases MMP Inhibitors Cell Cycle Regulatory Genes Cell Cycle Arrests and Checkpoint genes Negative Regulators of Cell Proliferation Positive Regulators of Cell Proliferation Growth Factors and Hormones Cytokines and Chemokines Receptors Pro-Apoptosis Anti-Apoptosis Transcription Factors Run 3 PCR Arrays for each sample to calculate FC (and p value) using the ∆∆Ct method Profiling genes by pathways and diseases - 27 - Sample & Assay Technologies
  28. 28. Scatter Plot Result ∆ Log 10 2-∆C(t) Breast Tumor (metastatic) UP REGULATED MMP9 MMP2 FGFR4 MMP7 ETV4 MMP3 MMP10 DOWN REGULATED ∆ Log 10 2-∆C(t) Breast Tumor (non-metastatic) Profiling genes by pathways and diseases - 28 - Scatter Plot Sample & Assay Technologies
  29. 29. MMP7 MMP9 MMP3 Log10 (P-value) Statistical Significance Volcano Plot Result ETV4 MMP10 MMP2 FGFR4 DOWN REGULATED UP REGULATED Log 2 Fold Change Profiling genes by pathways and diseases - 29 - Volcano Plot Sample & Assay Technologies
  30. 30. Seminar Overview Cancer Research Apoptosis What is Epigenetics? PCR Arrays for Gene Expression Profiling Tumor and Metastatic Tumor Cells Cell Cycle Inflammation ECM/Adhesion Neuroscience PCR Arrays for Epigenetic Applications Profiling miRNA Biomarkers in Colon Cancer Profiling DNA Methylation in Breast Cancer Cell lines and Normal Breast Tissue Profiling Chromatin Structure through Histone Modifications Signal Transduction Stem Cell/ Development Summary and Questions Toxicology/ Drug ADME The applications presented here are for research purposes. Not for diagnostic use. Profiling genes by pathways and diseases - 30 - Sample & Assay Technologies
  31. 31. What Is Micro RNA? Endogenously expressed small functional RNAs (~ 21-23 nt) . Regulate mRNA expression post-transcriptionally Primarily translation inhibition, but also mRNA degradation . Only partial sequence complementarity required One miRNA can regulate multiple mRNA One mRNA can be regulated by multiple miRNA Another layer of complexity for regulating gene expression . . Profiling genes by pathways and diseases - 31 - Sample & Assay Technologies
  32. 32. miRNAs as Biomarkers in Cancer Is there specific miRNAs expression differences in Human Colon tumors versus normal adjacent tissue? Normal Colon or Colon Tumor Tissue Extraction of total RNA QIAGEN miRNeasy Mini Kit miRNA Profiling genes by pathways and diseases - 32 - miScript miRNA PCR ARRAYS Sample & Assay Technologies
  33. 33. miRNA Biosynthetic Pathway Mature miRNA P Dicer miRNA pre-miRNA RISC miRNA-RISC Drosha P miRNA target recognition pri-miRNA Nucleus Translation Inhibition Message Turnover miRNA gene Profiling genes by pathways and diseases - 33 - Sample & Assay Technologies
  34. 34. miRNA Biosynthetic Pathway Mature miRNA P Dicer miRNA pre-miRNA RISC miRNA-RISC Drosha P miRNA target recognition pri-miRNA Nucleus Translation Inhibition Message Turnover miRNA gene Profiling genes by pathways and diseases - 34 - Sample & Assay Technologies
  35. 35. miScript miRNA PCR Arrays cDNA Synthesis 1 hours . Load Plates (Preferably with 8-Channel Pipettors) 2 minutes . Run 40 cycle qPCR Program 2 hours . Upload and Analyze Data 15 minutes . Profiling genes by pathways and diseases - 35 - Sample & Assay Technologies
  36. 36. Anatomy of a catalogued miRNA PCR Array miScript miRNA PCR Array Human miFinder (MIHS-001Z) 84 Pathway-Specific miRNAs . miR- miR- miR- miR- miR142-5p 16 142-3p 21 15a miR29b Let7a miR126 miR143 Let7b miR27a Let7f miR- miR9 26a miR24 miR- miR30e 181a miR- miR- miR29a 124 144 miR- miR30d 19b miR- miR122 22 miR- miR150 32 miR- miR- miR155 140-5p 125b miR- miR- miR141 92a 424 miR- miR17 191 miR- miR130a 20a miR- miR27b 26b miR146a miR- miR200c 99a miR- miR- miR19a 23a 30a Let7i Let7c miR101 Let7g miR425 miR- miR15b 28-5p miR- miR- miR18a 25 23b miR- miR302a 186 Let7d miR- miR30c 181b miR- miR223 320 miR374a miRNA isolation control n=2 . 6 “Housekeeping” snRNAs . miR93 miR106b . miR- miR- miR103 96 302b miR- miR29c 7 Let- miR- miR- miR- miR- miR7e 151-5p 374b 196b 140-3p 100 miR- miR- miR- miR- miR194 125a-5p423-5p 376c 195 SNORD SNORD SNORD SNORD SNORD RNU CelCel68 72 95 96A miR-39 miR-39 61 6-2 miR- miR- miR- miR222 28-3p 128a 302c MiRTC MiRTC PPC PPC miRNA Reverse Transcription Controls (miRTC) n=2 . Positive PCR Controls (PPC) n=2 . The miRNA PCR Arrays and reagents are intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of disease. Profiling genes by pathways and diseases - 36 - Sample & Assay Technologies
  37. 37. miScript miRNA RNA PCR Arrays: Available for Human, Mouse, Rat and Dog, Rhesus Macque miRNome (miRBase V16) Human Mouse Rat Dog miRNome (miRBase V16) 384 Well Format Human Mouse Rat Dog miFinder Human Mouse Rat Dog Rhesus Macaque miFinder 384HC Human Mouse Brain Cancer Human Mouse Rat Breast Cancer Human Mouse Rat Cancer PathwayFinder Human Mouse Rat Cell Differentiation & Development Human Mouse Rat Immunopathology Human Mouse Rat Inflammatory Response and Autoimmunity Human Mouse Rat Neurological Development & Disease Human Mouse Rat Ovarian Cancer Human Mouse Rat Prostate Cancer Human Mouse Rat Serum & Plasma Human Mouse Rat T-Cell & B-Cell Activation Human Mouse Rat miRNA QC Human Mouse Rat Dog Profiling genes by pathways and diseases - 37 - Sample & Assay Technologies
  38. 38. miRNAs as Biomarkers in Cancer Is there specific miRNAs expression differences in Human Colon tumors versus normal adjacent tissue? Normal Colon or Colon Tumor Tissue Extraction of total RNA QIAGEN miRNeasy Mini Kit miRNA Profiling genes by pathways and diseases - 38 - miScript Human CancerPathwayFinder miRNA PCR Array Sample & Assay Technologies
  39. 39. Colon Cancer Biomarkers miScript Human CancerPathwayfinder PCR Array: Many Cancer-Specific miRNA Are Up-regulated in the Colon Tumor. Profiling genes by pathways and diseases - 39 - Sample & Assay Technologies
  40. 40. Colon Cancer Biomarkers What about targets of the miRNAs? Is it possible to measure mRNA from the same sample? Profiling genes by pathways and diseases - 40 - Sample & Assay Technologies
  41. 41. Use PCR Arrays to Measure Pathway Specific Gene Expression Is there specific miRNAs expression differences in Human Colon tumors versus normal adjacent tissue? Normal Colon or Colon Tumor Tissue Extraction of total RNA QIAGEN miRNeasy Mini Kit RT2 Profiler PCR ARRAYS Archive “large” mRNA fraction For use on PCR Arrays Profiling genes by pathways and diseases miRNA - 41 - miScript miRNA ARRAYS Sample & Assay Technologies
  42. 42. Seminar Overview Cancer Research Apoptosis What is Epigenetics? PCR Arrays for Gene Expression Profiling Tumor and Metastatic Tumor Cells Cell Cycle Inflammation ECM/Adhesion PCR Arrays for Epigenetic Applications Profiling miRNA Biomarkers in Colon Cancer Profiling DNA Methylation in Breast Cancer Cell lines and Normal Breast Tissue Profiling Histone Modification on CDKN1A Neuroscience Summary and Questions Signal Transduction Stem Cell/ Development Toxicology/ Drug ADME The applications presented here are for research purposes. Not for diagnostic use. Profiling genes by pathways and diseases - 42 - Sample & Assay Technologies
  43. 43. CpG Islands and Methylation • CpG islands: CpG-rich regions in human gene promoters – Most CpG dinucleotides are methylated outside of promoter regions – Healthy/normal cells: rarely methylated – Cancer cells: hypermethylated, leading to transcriptional silencing Normal 1 2 3 1 2 3 Expression Promoter Region Cancer – Important genes (i.e. tumor suppressors) are silenced – No Expression All cellular pathways related to cancer can be affected Adapted from Herman, J. and Baylin, S. N. Engl. J. Med. 2003 Profiling genes by pathways and diseases - 43 - Sample & Assay Technologies
  44. 44. Background DNA Methylation Biological Importance Role in normal development and also disease progression Potentially silences critical tumor suppressors Involves all cellular pathways Stable/Covalent modificaton which can be detected in heterogeneous fluids Promising biomarker for early detection and predicting prognosis . Profiling genes by pathways and diseases - 44 - Sample & Assay Technologies
  45. 45. DNA Methylation and Transcriptional Repression Blood, 93:4059-4070, 1999 Profiling genes by pathways and diseases - 45 - Sample & Assay Technologies
  46. 46. Methylation in Oncogenesis: Study Design What are the 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) Bioinformatics text mining: Custom PCR Arrays: genes hypermethylated in Breast Cancer with high frequencies Profiling genes by pathways and diseases - 46 - Sample & Assay Technologies
  47. 47. EpiTect Methy lI PCR Arrays (24 and 96 gene panels) 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 Breast Cancer Pathway-Focused Methyl qPCR Arrays • • • • • • • • • • • • • Epithelial to Mesenchymal Transition Gastric Cancer Liver Cancer Lung Cancer Prostate Cancer Colon Cancer Leukemia & Lymphoma Tumor Suppressor Genes Apoptosis DNA Repair Stem Cell Transcription Factors Inflammatory Response T Cell Activation Cytokine Production Tumor Suppressor Genes Homeobox (HOX) Genes Polycomb (PcG) Genes Cell Cycle Toll-Like Receptor Signaling TGF-Beta/BMP Signaling Wnt Signaling Custom Arrays and Custom Assays Available for most CpG island-containing promoter in the human, mouse, or rat genome. http://www.sabiosciences.com/dna_methylation_custom_PCRarray.php Profiling genes by pathways and diseases - 47 - Sample & Assay Technologies
  48. 48. DNA Methylation PCR Array Protocol Overview (0.5 – 4 ug of Genomic DNA) 24 gene- Signature Panel Profiling genes by pathways and diseases - 48 - Sample & Assay Technologies
  49. 49. DNA Methylation PCR Array Protocol Overview (0.5 – 4 ug of Genomic DNA) 22 gene- Signature Panel Profiling genes by pathways and diseases - 49 - Sample & Assay Technologies
  50. 50. DNA Methylation PCR Array Protocol Overview (0.5 – 4 ug of Genomic DNA) Data Analysis using downloadable Excel Templates Accurate calculates ∆Ct values and converts to % 22 gene- Signature Panel Profiling genes by pathways and diseases - 50 - Sample & Assay Technologies
  51. 51. EpiTect Methyl II Biomarker Discovery EpiTect Methtl II PCR Arrays can be effectively used to identify novel hypermethylated biomarkers. Profiling genes by pathways and diseases - 51 - Sample & Assay Technologies
  52. 52. EpiTect Methyl II Biomarker Discovery EpiTect Methtl II PCR Arrays can be effectively used to identify novel hypermethylated biomarkers. Measure gene expression using cataloged or custom RT2 Profiler PCR Arrays Use Epitect Pyrosequencing Assays to quantitatively identify Methylation at the single nucleotide level. Profiling genes by pathways and diseases - 52 - Sample & Assay Technologies
  53. 53. Seminar Overview Cancer Research Apoptosis What is Epigenetics? PCR Arrays for Gene Expression Profiling Tumor and Metastatic Tumor Cells Cell Cycle Inflammation ECM/Adhesion PCR Arrays for Epigenetic Applications Profiling miRNA Biomarkers in Colon Cancer Profiling DNA Methylation in Breast Cancer Cell lines and Normal Breast Tissue Profiling Histone Modification on CDKN1A Neuroscience Signal Transduction Summary and Questions Stem Cell/ Development Toxicology/ Drug ADME The applications presented here are for research purposes. Not for diagnostic use. Profiling genes by pathways and diseases - 53 - Sample & Assay Technologies
  54. 54. What IS ChIP? What is ChIP? Chromatin Immuno-Precipitation Method to examine in vivo protein-DNA interactions Applications: Transcription Factors, Modified Histones, Other nuclear factors . . . ChIP-qPCR is Chromatin Immunoprecipitation with qPCR Detection and Quantification Profiling genes by pathways and diseases - 54 - Sample & Assay Technologies
  55. 55. 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? Use Custom ChIP PCR Array for multiple “tiles” within 1 gene Or use individual ChIP qPCR Assay What is the chromatin structure? Is it open or closed? (histone modifications) Example: Can p53 bind to this site? Use Cataloged Array for pathway (84 genes looking at +1 “tile” Or Use individual qPCR Assay/ Custom PCR Array Profiling genes by pathways and diseases - 55 - Sample & Assay Technologies
  56. 56. ChIP PCR Arrays: 2 different applications and Layouts Profiling 1 gene with 30 tiles What is the chromatin structure for the entire promoter region Profiling 1 tile (+1 tile) for 84 genes in a Pathway •Tumor Suppressor & Oncogenes •Cancer Drug Targets •DNA Repair •Cell Cycle •Stem Cell Transcription Factors •Inflammatory Response •T Hellper Cell Differentiation •Polycomb & Trithorax Target Genes •Polycomb & Trithorax Complexes •Homeobox (HOX) Genes Profiling genes by pathways and diseases - 56 - Sample & Assay Technologies
  57. 57. 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 Profiling genes by pathways and diseases - 57 - Sample & Assay Technologies
  58. 58. 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 . . . . Profiling genes by pathways and diseases - 58 - Sample & Assay Technologies
  59. 59. qPCR Assay Design: Tiling Scheme for Pathway Catalogued Arrays 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) 84 Genes with only +1 Tile Tiles +1kb relative to each TSS for 84 genes in a pathway Quality Controlled for Specificity & Amplification Efficiency . . Use the +1kb tile as a surrogate for promoter region to know transcriptional status Use many genes to understand pathway Correlate with gene expression . . . Profiling genes by pathways and diseases - 59 - Sample & Assay Technologies
  60. 60. Modified Histone Distribution Across a Promoter Custom ChIP PCR Array for Entire CDKN1A Gene 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. Profiling genes by pathways and diseases - 60 - Sample & Assay Technologies
  61. 61. Seminar Overview Cancer Research Apoptosis What is Epigenetics? PCR Arrays for Gene Expression Profiling Tumor and Metastatic Tumor Cells Cell Cycle Inflammation ECM/Adhesion PCR Arrays for Epigenetic Applications Profiling miRNA Biomarkers in Colon Cancer Profiling DNA Methylation in Breast Cancer Cell lines and Normal Breast Tissue Profiling Histone Modification on CDKN1A Neuroscience Signal Transduction Summary and Questions Stem Cell/ Development Toxicology/ Drug ADME The applications presented here are for research purposes. Not for diagnostic use. Profiling genes by pathways and diseases - 61 - Sample & Assay Technologies
  62. 62. PCR Array Summary Complete System (From Sample to Fold Change) Five-component system: Sample Isolation, PCR arrays, First strand (OR DNA RE Kit), master mix, and data analysis software . Breadth of Content CATALOGED Pathways or customizable Gene Expression MicroRNA Expression DNA Methylation Chromatin Immunoprecipitation (histone modifications/TF binding) Validated Real-Time PCR Performance Wet-Bench tested qPCR Assays for Sensitivity, Specificity, and Amplification Efficiency on every qPCR manufactured. Final Benefit to Researcher: . . Platform technology for any qPCR machine in any laboratory Profiling genes by pathways and diseases - 62 - Sample & Assay Technologies
  63. 63. Need More Info? Product Support Tools www.sabiosciences.com Global Technical Support Email: Support@SABiosciences.com Profiling genes by pathways and diseases - 63 - Sample & Assay Technologies
  64. 64. Thank you for attending! Questions, Comments, Suggestions, Concerns? Samuel.Rulli@QIAGEN.com Questions, Comments, Concerns? US Applications Support support@sabiosciences.com Profiling genes by pathways and diseases Questions, Comments, Concerns? Global Applications Support SABio@qiagen.com - 64 - Sample & Assay Technologies
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