Upcoming Webinars. Profiling genes by pathways and diseases. Sample & Assay Technologies -1-

Transcription

1 Upcoming Webinars -1-

2 Keep up to date: Follow Pathway focused biology on Facebook Latest information on, pathway focused research and demos. -2-

3 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. -3-

4 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. -4-

5 Seminar Overview Cancer Research Apoptosis Cell Cycle Inflammation ECM/Adhesion Neuroscience Signal Transduction What is Epigenetics? PCR Arrays for Gene Expression Profiling Tumor and Metastatic Tumor Cells 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. -5-

6 Seminar Overview Cancer Research Apoptosis Cell Cycle Inflammation ECM/Adhesion Neuroscience Signal Transduction What is Epigenetics? PCR Arrays for Gene Expression Profiling Tumor and Metastatic Tumor Cells 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. -6-

7 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 -7-

8 Current View of Gene Expression Activated Transcription Factors RNAi: mirna Protein A NF B p53 + Transcription Initiation Complex mrna A Me p53 BS Histones AC AC Me NF B BS Histones Me Me Me Me Me Me Me TSS (+1) Structural Gene A Gene Expression is Complex with multiple layers of control -8-

9 Multiple Epigenetic mechanisms influence Gene Expression Activated Transcription Factors RNAi: mirna NF B p53 Transcription Initiation Complex mrna A Me p53 BS Histones AC AC Me NF B BS Histones Me Me Me Me Me Me Me TSS (+1) Structural Gene A Gene Expression is Complex with multiple layers of control -9-

10 PCR Arrays for Gene Expression and Epigenetic Regulation Activated Transcription Factors RNAi: mirna Protein A NF B p53 Transcription Initiation Complex mrna A Me p53 BS Histones AC AC Me NF B BS Histones Me Me Me Me Me Me Me TSS (+1) Structural Gene A Gene Expression is Complex with multiple layers of control -10-

11 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? -11-

12 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 -12-

13 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. RT 2 PCR Arrays (96, 384 and Rotorgene Q formats) 3. RT 2 First Strand kit** 4. RT 2 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 -13-

14 Seminar Overview Cancer Research Apoptosis Cell Cycle Inflammation ECM/Adhesion Neuroscience Signal Transduction What is Epigenetics? PCR Arrays for Gene Expression Profiling Tumor and Metastatic Tumor Cells 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. -14-

15 PCR Arrays for Gene Expression Cancer Research Research Question: What are the gene expression differences between a metastatic and non-metastatic Breast Tumor? -15-

16 Anatomy of a RT 2 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) -16-

17 Anatomy of a RT 2 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) -17-

18 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 -18-

19 How RT 2 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 -19-

20 How RT 2 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 -20-

21 How RT 2 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 -21-

22 How RT 2 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 -22-

23 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 Also available as downloadable Excel Volcano templates Plot -23-

24 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 -24-

25 PCR Arrays for All Biomedical Researchers ~ 100 different pathways for Human, Mouse, Rat, Monkey and Fly. Cancer and Apoptosis Apoptosis Cell Cycle Human mirna Array (NEW!) Breast Cancer & Estrogen Receptor Tumor Metastasis Cancer PathwayFinder Angiogenesis Cancer Drug Resistance Signal Transduction Signal Transduction PathwayFinder NFkB Signaling Jak / Stat Signaling DNA Damage Signaling Insulin Signaling MAP Kinase Signaling camp / Calcium Signaling p53 Signaling Cytokines & Inflammation Inflammatory Cytokines Th17 for Inflammation (NEW!) Common Cytokines Chemokines NF- B Signaling Pathway Th1-Th2-Th3 TNF Ligands Toll-like Receptors Toxicology & Drug Metabolism Drug Metabolism Drug Phase I Enzymes Drug Transporters Oxidative Stress Stress & Toxicity Other Diseases Atherosclerosis Diabetes Complete PCR Array List Development & Stem Cells Stem Cells Wnt Signaling Notch Signaling TGF / BMP Signaling Endothelial Cell Biology Osteogenesis Growth Factors ECM & Adhesion Neuroscience Neuroscience Ion Channels Neurotransmitter Receptors Neurotrophins & Receptors Neurogenesis and Neural Stem Cell Custom PCR Arrays 96-Well Custom Arrays 384-Well Custom Arrays -25-

26 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 -26-

27 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 -27-

28 PCR Arrays for All Biomedical Researchers ~ 100 different pathways for Human, Mouse, Rat, Monkey and Fly. Cancer Research 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 Research Question: What are the gene expression differences between a metastatic and non-metastatic Breast Tumor? -28-

29 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 -29-

30 Scatter Plot Result UP REGULATED Log C(t) Breast Tumor (metastatic) FGFR4 MMP9 MMP2 MMP7 ETV4 MMP3 MMP10 Log C(t) Breast Tumor (non-metastatic) DOWN REGULATED Scatter Plot -30-

31 Volcano Plot Result Statistical Significance Log 10 (P-value) MMP7 MMP3 ETV4 MMP2 MMP10 MMP9 FGFR4 DOWN REGULATED UP REGULATED Log 2 Fold Change Volcano Plot -31-

32 Seminar Overview Cancer Research Apoptosis Cell Cycle Inflammation ECM/Adhesion Neuroscience Signal Transduction Stem Cell/ Development What is Epigenetics? PCR Arrays for Gene Expression Profiling Tumor and Metastatic Tumor Cells 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 Toxicology/ Drug ADME The applications presented here are for research purposes. Not for diagnostic use. -32-

33 What Is Micro RNA?. Endogenously expressed small functional RNAs (~ 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. -33-

34 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 miscript mirna PCR ARRAYS -34-

35 mirna Biosynthetic Pathway Dicer Mature mirna P pre-mirna mirna Drosha mirna-risc RISC P pri-mirna mirna target recognition Nucleus mirna gene Translation Inhibition Message Turnover -35-

36 mirna Biosynthetic Pathway Dicer Mature mirna P pre-mirna mirna Drosha mirna-risc RISC P pri-mirna mirna target recognition Nucleus mirna gene Translation Inhibition Message Turnover -36-

37 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 -37-

38 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 -38-

39 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 miscript Human CancerPathwayFinder mirna PCR Array -39-

40 Colon Cancer Biomarkers miscript Human CancerPathwayfinder PCR Array: Many Cancer-Specific mirna Are Up-regulated in the Colon Tumor. -40-

41 Colon Cancer Biomarkers What about targets of the mirnas? Is it possible to measure mrna from the same sample? -41-

42 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 RT 2 Profiler PCR ARRAYS Archive large mrna fraction For use on PCR Arrays mirna QIAGEN mirneasy Mini Kit miscript mirna ARRAYS -42-

43 Seminar Overview Cancer Research Apoptosis Cell Cycle Inflammation ECM/Adhesion Neuroscience Signal Transduction What is Epigenetics? PCR Arrays for Gene Expression Profiling Tumor and Metastatic Tumor Cells 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 Summary and Questions Stem Cell/ Development Toxicology/ Drug ADME The applications presented here are for research purposes. Not for diagnostic use. -43-

44 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 Promoter Region Expression Cancer No Expression Important genes (i.e. tumor suppressors) are silenced All cellular pathways related to cancer can be affected Adapted from Herman, J. and Baylin, S. N. Engl. J. Med

45 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 -45-

46 DNA Methylation and Transcriptional Repression Blood, 93: ,

47 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 -47-

48 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 Epithelial to Mesenchymal Transition Gastric Cancer Liver Cancer Lung Cancer Prostate Cancer Colon Cancer Leukemia & Lymphoma Tumor Suppressor Genes Pathway-Focused Methyl qpcr Arrays 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

49 DNA Methylation PCR Array Protocol Overview (0.5 4 ug of Genomic DNA) 24 gene- Signature Panel -49-

50 DNA Methylation PCR Array Protocol Overview (0.5 4 ug of Genomic DNA) 22 gene- Signature Panel -50-

51 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 -51-

52 EpiTect Methyl II Biomarker Discovery EpiTect Methtl II PCR Arrays can be effectively used to identify novel hypermethylated biomarkers. -52-

53 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 RT 2 Profiler PCR Arrays Use Epitect Pyrosequencing Assays to quantitatively identify Methylation at the single nucleotide level. -53-

54 Seminar Overview Cancer Research Apoptosis Cell Cycle Inflammation ECM/Adhesion Neuroscience Signal Transduction What is Epigenetics? PCR Arrays for Gene Expression Profiling Tumor and Metastatic Tumor Cells 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 Summary and Questions Stem Cell/ Development Toxicology/ Drug ADME The applications presented here are for research purposes. Not for diagnostic use. -54-

55 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 -55-

56 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 -56-

57 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 -57-

58 Chromatin Immunoprecipitation Workflow Stimulus X-Link Lyse Fragment X X IP X Reverse X-Link Purify DNA Input Anti-TF NIS Real-Time PCR Primers ChIP-qPCR -58-

59 qpcr Assay Design: Tiling Scheme TSS (+1) Structural Gene mrna Distal Promoter Proximal Promoter gdna 1 kb 1 kb 1 kb 1 kb 1 kb. qpcr Assay & Amplicon 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 -59-

60 qpcr Assay Design: Tiling Scheme for Pathway Catalogued Arrays TSS (+1) Structural Gene mrna Distal Promoter Proximal Promoter gdna 1 kb 1 kb 1 kb 1 kb 1 kb. qpcr Assay & Amplicon 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 -60-

61 Modified Histone Distribution Across a Promoter -1 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. -61-

62 Seminar Overview Cancer Research Apoptosis Cell Cycle Inflammation ECM/Adhesion Neuroscience Signal Transduction What is Epigenetics? PCR Arrays for Gene Expression Profiling Tumor and Metastatic Tumor Cells 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 Summary and Questions Stem Cell/ Development Toxicology/ Drug ADME The applications presented here are for research purposes. Not for diagnostic use. -62-

63 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 -63-

64 Need More Info? Product Support Tools Global Technical Support

65 Thank you for attending! Copy of the slides? Just click Yes! in the survey and leave a valid address. Questions, Comments, Suggestions, Concerns? Samuel.Rulli@QIAGEN.com Questions, Comments, Concerns? US Applications Support support@sabiosciences.com Questions, Comments, Concerns? Global Applications Support SABio@qiagen.com -65-