Pathway-Powered PCR Arrays

Discovery & Validation of Biomarkers to Monitor
Genotoxicity by Gene Expression Profiling

Bil...
Table of Contents

1.

Introduction to Genotoxicity

2.

Development of Gene-Based In Vitro Genotoxic
Biomarkers Using PCR...
Drug Development At A Glance
7 millions compounds screened
1000 hits

Efficacy and Safety

12 candidates
6 candidates

1 p...
Why Toxicity Is Concerned
Reasons For Failure During Drug Discovery & Development Process

The inability to accurately pre...
Current Hot Spots in Toxicity Risk Assessment

Hepato
Toxicity
Pulmonary
Toxicity

Neuro
Toxicity

Geno
Toxicity

Cardio
T...
Classification of Carcinogens

Genotoxic carcinogens:
Cause irreversible genetic damage or mutation by binding to DNA.
Dir...
Current Testing Battery For Genotoxicity
Bacterial mutagenesis (Ames Test)
In vitro mammalian mutagenesis
In vitro chromos...
Gene Expression Regulates Biology

All require molecular signaling for action

For Internal Use Only

-8-

Sample & Assay ...
Gene Expression And Genotoxicity

Gene expression profiles should be profoundly different between
genotoxic and non-genoto...
Biological Markers Define Processes

Angiogenesis

Inflammation

P53
MDM2
Cyclins
CDKs

VEGF
bFGF
ANGPT
PDGF

IL8
TLR
IFNγ...
Complete Biological Story
Built on Pathway / Network Analysis

Angiogenesis

Inflammation

Pathway

Cell cycle

For Intern...
Differential Gene Expression Analysis

Q: How to Assess the Expression of
Different mRNAs in a Sample
involved in a Pathwa...
Table of Contents

1.

Introduction to Genotoxicity

2.

Development of Gene-Based In Vitro Genotoxic
Biomarkers Using PCR...
Principles of qRT-PCR in PCR Arrays

Real-Time PCR
• Amplify & simultaneously quantify target DNA

Reverse Transcription R...
Anatomy of a PCR Array

•

84 Pathway-Specific Genes
of Interest

•

5 Housekeeping Genes
– Normalization

•

Genomic DNA ...
Exquisite Specificity

• Assays on Human TGFβ &
BMP Signaling Pathway
PCR Array used to analyze
BMP genes in Universal
Ref...
Uniform Amplification Efficiency

Representative set of 500 out of > 4,000 assays used in PCR Arrays
CONCLUSION: Accurate ...
High Reproducibility
Reproducibility Among Different Instruments

Reproducibility Among Different Users

Human Drug Metabo...
Definition of “PATHWAY-FOCUSED” Analysis

“Pathway-Focused” describes examination of:
• biological signaling events,
• cla...
PCR Arrays for ALL Biomedical Researchers
Cancer and Apoptosis

Cytokines & Inflammation

Development & Stem Cells

Apopto...
How Genes on PCR Arrays Are Selected
Biologically relevant gene content
• Not simply biochemical pathways or kinase
cascad...
Development of Genotoxic Biomarker
Compound Selection

For Internal Use Only

- 22 -

Sample & Assay Technologies
Development of Genotoxic Biomarker
Study Design

Cells:

.

HepG2 cells: 2 X 105 cells/ml at seeding.
96-well plate for cy...
How RT2 Profiler PCR Arrays Work
Experiment:
QIAcube

Non-Genotoxic
QIAxcel
TL II

Genotoxic
RNA Isolation

Automated
Solu...
RT2 Profiler™ PCR Array Data Analysis
FREE Complete & Easy Analysis with Web-/Excel-Based Software

• Gene Content for eac...
Expression Profiles of 11 Signature Genes

For Internal Use Only

- 26 -

Sample & Assay Technologies
Gene Signatures Define Compound Class

For Internal Use Only

- 27 -

Sample & Assay Technologies
Expression Profiles From Different Modes of Action

For Internal Use Only

- 28 -

Sample & Assay Technologies
Summary 1

We described the concept of pathway focused gene expression
analysis using RT2 Profiler PCR Arrays.
.

We ident...
Table of Contents

1.

Introduction to Genotoxicity

2.

Development of Gene-Based In Vitro Genotoxic
Biomarkers Using PCR...
miRNA Biology

For Internal Use Only

- 31 -

Sample & Assay Technologies
miRNAs as Master Regulators

For Internal Use Only

- 32 -

Sample & Assay Technologies
miScript miRNA PCR Array System
miRNA Pathway & Whole miRNome Profiling

Complete Workflow Solution for miRNA Expression A...
miRNA Profiling After Exposure To Genotoxic Carcinogen
Study Design

Goal: explore miRNAs as potential biomarkers for geno...
Temporal Changes In Differentially Expressed miRNA

BMC Genomics 2010, 11:609
For Internal Use Only

- 35 -

Sample & Assa...
Clustering Of Differentially Expressed miRNAs

mir-34 family

BMC Genomics 2010, 11:609
For Internal Use Only

- 36 -

Sam...
Temporal Changes Of mir-34 Family
TaqMan individual

PCR array
16

16

8

*

*

8

*

*

4

mmu-miR-34a

mmu-miR-34a

*

4...
Top 10 Biological Functions Implicated By Differentially
Expressed miRNA
Differentially expressed miRNA at day 7 and 15
Ta...
Summary 2

We introduced the concept of miRNA and miRNA profiling using
miScript PCR Arrays.

.

Exposure to carcinogen (E...
Take Home Message

Gene expression not only regulates biology, but also serves as
a surrogate to monitor biology.

.

Path...
Pathway-Focused Expression Analysis
Complete Experimental Systems: Sample Prep through Data Analysis

For Internal Use Onl...
Thank You for Attending!

Call 1-888-503-3187 to order
Email: support@SABioscience.com

For Internal Use Only

- 42 -

Sam...
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Biomarker for genotoxicity 2013

  1. 1. Pathway-Powered PCR Arrays Discovery & Validation of Biomarkers to Monitor Genotoxicity by Gene Expression Profiling Bill Wang, Ph.D. QIAGEN For Internal Use Only -1- Sample & Assay Technologies
  2. 2. Table of Contents 1. Introduction to Genotoxicity 2. Development of Gene-Based In Vitro Genotoxic Biomarkers Using PCR Arrays • • • 3. Introduction to RT2 Profiler PCR Array Experimental Design Results Identifying miRNA-Based In Vivo Biomarkers to Monitor Genotoxicity in Mouse • • • Introduction to miScript PCR Array Experimental Design Results For Internal Use Only -2- Sample & Assay Technologies
  3. 3. Drug Development At A Glance 7 millions compounds screened 1000 hits Efficacy and Safety 12 candidates 6 candidates 1 product Early Discovery Phase Exploratory Development Phase I, II 0 Idea 5 10 15yr 12 to 24 years Cost: ~ $900 million For Internal Use Only Clinical Development Phase III -3- Marketed Drug Sample & Assay Technologies
  4. 4. Why Toxicity Is Concerned Reasons For Failure During Drug Discovery & Development Process The inability to accurately predict toxicity early in drug development cost the pharmaceutical industry billions and approximately one-third the cost of all drug failures. FAIL IT EARLIER, FAIL IT CHEAPER ! Drug Discovery Today 2007 12:289-91 For Internal Use Only -4- Sample & Assay Technologies
  5. 5. Current Hot Spots in Toxicity Risk Assessment Hepato Toxicity Pulmonary Toxicity Neuro Toxicity Geno Toxicity Cardio Toxicity Renal Toxicity For Internal Use Only Immuno Toxicity -5- Sample & Assay Technologies
  6. 6. Classification of Carcinogens Genotoxic carcinogens: Cause irreversible genetic damage or mutation by binding to DNA. Direct: DNA cross-linking. DNA adduct formation. Indirect: Interfere with the function of proteins that are involved in DNA replication or chromosome stability. Non-genotoxic carcinogens: Don’t directly affect DNA but act in other ways to initiate, promote or aggravate tumor development: multiple and diverse mechanisms. For Internal Use Only -6- Sample & Assay Technologies
  7. 7. Current Testing Battery For Genotoxicity Bacterial mutagenesis (Ames Test) In vitro mammalian mutagenesis In vitro chromosome aberration assay In vitro micronucleus assay In vivo chromosome stability assay 2 year rodent carcinogenicity test. Accuracy: 38%, with high false positive rate. High toxic dose (50-80% growth inhibition) used --- low sensitivity Difficulty at prediction of carcinogenicity at low doses to which human subjects are usually exposed. For non-genotoxic carcinogens: no suitable model available. For Internal Use Only -7- Sample & Assay Technologies
  8. 8. Gene Expression Regulates Biology All require molecular signaling for action For Internal Use Only -8- Sample & Assay Technologies
  9. 9. Gene Expression And Genotoxicity Gene expression profiles should be profoundly different between genotoxic and non-genotoxic carcinogens. Gene expression profiles should be able to discriminate among compounds having different mechanisms. The toxicity of unknown compounds can be predicted by comparison of their molecular fingerprints with those obtained with compounds of known toxicity. For Internal Use Only -9- Sample & Assay Technologies
  10. 10. Biological Markers Define Processes Angiogenesis Inflammation P53 MDM2 Cyclins CDKs VEGF bFGF ANGPT PDGF IL8 TLR IFNγ TNFβ Individual participants Cell cycle For Internal Use Only - 10 - Sample & Assay Technologies
  11. 11. Complete Biological Story Built on Pathway / Network Analysis Angiogenesis Inflammation Pathway Cell cycle For Internal Use Only - 11 - Sample & Assay Technologies
  12. 12. Differential Gene Expression Analysis Q: How to Assess the Expression of Different mRNAs in a Sample involved in a Pathway and Compare it across Multiple Conditions? A: For Internal Use Only - 12 - Sample & Assay Technologies
  13. 13. Table of Contents 1. Introduction to Genotoxicity 2. Development of Gene-Based In Vitro Genotoxic Biomarkers Using PCR Arrays • • • 3. Introduction to RT2 Profiler PCR Array Experimental Design Results Identifying miRNA-Based In Vivo Biomarkers to Monitor Genotoxicity in Mouse • • • Introduction to miScript PCR Array Experimental Design Results For Internal Use Only - 13 - Sample & Assay Technologies
  14. 14. Principles of qRT-PCR in PCR Arrays Real-Time PCR • Amplify & simultaneously quantify target DNA Reverse Transcription Real-Time PCR • Amplify & simultaneously quantify messenger RNA (mRNA) Ct Values • Threshold Cycle For Internal Use Only - 14 - Sample & Assay Technologies
  15. 15. Anatomy of a PCR Array • 84 Pathway-Specific Genes of Interest • 5 Housekeeping Genes – Normalization • Genomic DNA Contamination Detection Assay • Reverse Transcription Controls (RTC) n=3 – Spike-in control • Positive PCR Controls (PPC) n=3 Sample & Assay Technologies
  16. 16. Exquisite Specificity • Assays on Human TGFβ & BMP Signaling Pathway PCR Array used to analyze BMP genes in Universal Reference RNA. • Single peak dissociation curves • Single gel bands of predicted size • High specificity for genes difficult to design specific primers for. Conclusion: Each well in a PCR Array detects a single genespecific product. For Internal Use Only - 16 - Sample & Assay Technologies
  17. 17. Uniform Amplification Efficiency Representative set of 500 out of > 4,000 assays used in PCR Arrays CONCLUSION: Accurate and reliable ∆∆Ct results are guaranteed. For Internal Use Only - 17 - Sample & Assay Technologies
  18. 18. High Reproducibility Reproducibility Among Different Instruments Reproducibility Among Different Users Human Drug Metabolism PCR Array in Universal Reference RNA Conclusion: Same raw threshold cycle data can be obtained for the same samples even from different end users at different times or using different instruments. For Internal Use Only - 18 - Sample & Assay Technologies
  19. 19. Definition of “PATHWAY-FOCUSED” Analysis “Pathway-Focused” describes examination of: • biological signaling events, • classes of genes, or • those genes related to diseases. PCR Arrays allow for easy & simultaneous analysis of a PathwayFocused set of genes. For Internal Use Only - 19 - Sample & Assay Technologies
  20. 20. PCR Arrays for ALL Biomedical Researchers Cancer and Apoptosis Cytokines & Inflammation Development & Stem Cells Apoptosis Inflammatory Cytokines Stem Cells Cell Cycle Th17 for Inflammation WNT Signaling / Notch Signaling Human miRNA Array Common Cytokines / Chemokines Terminal Differentiation Markers Breast Cancer & Estrogen Receptor Inflammasomes TGFβ / BMP Signaling Tumor Metastasis NF-kB Signaling Pathway Endothelial Cell Biology Epithelial-to-Mesenchymal Transition 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 / Drug Transporters Neuroscience Ion Channels NFkB Signaling Drug Phase I Enzymes Neurotransmitter Receptors Jak / Stat Signaling Molecular Toxicology PathwayFinder 384HT Neurotrophins & Receptors DNA Damage Signaling Oxidative Stress Neurogenesis and Neural Stem Cell Insulin Signaling Stress & Toxicity Parkinson’s Disease MAP Kinase Signaling Other Diseases cAMP / Calcium Signaling Atherosclerosis 96-Well, 384-Well Plate p53 Signaling Diabetes 100-Well Disc, 96x96 Chip Custom PCR Arrays (H/M/R/Q/D/F/P/B) ** Over 150 Pathway- Powered PCR Arrays Available** Sample & Assay Technologies
  21. 21. 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 gathered from 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 Human DNA Damage Signaling Pathway RT2 PCR Array • Genes involved in DNA damage, repair, cell cycle control, and apoptosis. • Specific feedback from thought leaders Custom PCR Array • Additional tentative signature genes derived from various microarray studies (GEO) in the context of genotoxicity. For Internal Use Only - 21 - Genes in Signaling Pathways Genes with High Relevance Sample & Assay Technologies
  22. 22. Development of Genotoxic Biomarker Compound Selection For Internal Use Only - 22 - Sample & Assay Technologies
  23. 23. Development of Genotoxic Biomarker Study Design Cells: . HepG2 cells: 2 X 105 cells/ml at seeding. 96-well plate for cytotoxicity assays --- 3 to 5 biological replicates. 6-well plate for RNA isolation --- 4 biological replicates. . . . Dose: . IC20 - low dose but substantial to trigger gene expression changes. . Time: . 24 hr – to avoid generic stress responses often observed at shorter incubation time (e.g. 3 hr or 6 hr). . RNA Isolation and PCR Array Analysis: . RT2 PCR Array protocols. . For Internal Use Only - 23 - Sample & Assay Technologies
  24. 24. How RT2 Profiler PCR Arrays Work Experiment: QIAcube Non-Genotoxic QIAxcel TL II Genotoxic RNA Isolation Automated Solutions RNA Isolation (RNeasy) RNase-Free DNase Treatment Cells & Tissues RNA (25ng- 5ug)* gDNA Elimination Step Artificial RNA Spike In (RTC) First Strand cDNA Synthesis ~45 minutes 1st strand cDNA RGQ + SYBR Green Master Mix Real-time PCR Detection of 89 Gene-specific Amplification on RT2 Profiler PCR Array 2 hours 15 minutes For Internal Use Only *RT2 PreAMP for 1 ng Upload & Analyze Data - 24 - Sample & Assay Technologies
  25. 25. RT2 Profiler™ PCR Array Data Analysis FREE Complete & Easy Analysis with Web-/Excel-Based Software • Gene Content for each Pathway is Pre-Loaded • Custom Arrays: Gene List is all you need • From Raw Ct Values to Fold Change Results in Multiple Analysis Formats Volcano Plot For Internal Use Only Scatter Plot - 25 - Clustergram 3-D Histogram Sample & Assay Technologies
  26. 26. Expression Profiles of 11 Signature Genes For Internal Use Only - 26 - Sample & Assay Technologies
  27. 27. Gene Signatures Define Compound Class For Internal Use Only - 27 - Sample & Assay Technologies
  28. 28. Expression Profiles From Different Modes of Action For Internal Use Only - 28 - Sample & Assay Technologies
  29. 29. Summary 1 We described the concept of pathway focused gene expression analysis using RT2 Profiler PCR Arrays. . We identified 11 genes in DNA damage repair and p53 pathways as a classifier for genotoxic and non-genotoxic compounds. . Genotoxic compounds with different modes of action elicit distinct gene expression profiles. . For Internal Use Only - 29 - Sample & Assay Technologies
  30. 30. Table of Contents 1. Introduction to Genotoxicity 2. Development of Gene-Based In Vitro Genotoxic Biomarkers Using PCR Arrays • • • 3. Introduction to RT2 Profiler PCR Array Experimental Design Results Identifying miRNA-Based In Vivo Biomarkers to Monitor Genotoxicity in Mouse • • • Introduction to miScript PCR Array Experimental Design Results For Internal Use Only - 30 - Sample & Assay Technologies
  31. 31. miRNA Biology For Internal Use Only - 31 - Sample & Assay Technologies
  32. 32. miRNAs as Master Regulators For Internal Use Only - 32 - Sample & Assay Technologies
  33. 33. miScript miRNA PCR Array System miRNA Pathway & Whole miRNome Profiling Complete Workflow Solution for miRNA Expression Analysis . miRNA Isolation & cDNA Synthesis Universal Primer to 40 cycle qPCR Accurate Results Easy Data Analysis PCR Arrays: Pathways and Genomes Serum (Circulating Disease) – New! Neurological Development & Disease Brain Cancer Cell Differentiation & Development Immunopathology Inflammation miFinder Whole Genome (miRNome): Human, Mouse, Rat, Dog . For Internal Use Only - 33 - Sample & Assay Technologies
  34. 34. miRNA Profiling After Exposure To Genotoxic Carcinogen Study Design Goal: explore miRNAs as potential biomarkers for genotoxicity Subject: 6-month old female mice Compound: N-ethyl-N-nitrosourea (ENU) 120mg/kg body weight Time course: . . . . DMSO 1 30 Mice ENU Days 1 3 7 15 30 120 Sample source: miRNA from liver Analysis: miRNA PCR Array for 384 most expressed miRNAs . . For Internal Use Only - 34 - Sample & Assay Technologies
  35. 35. Temporal Changes In Differentially Expressed miRNA BMC Genomics 2010, 11:609 For Internal Use Only - 35 - Sample & Assay Technologies
  36. 36. Clustering Of Differentially Expressed miRNAs mir-34 family BMC Genomics 2010, 11:609 For Internal Use Only - 36 - Sample & Assay Technologies
  37. 37. Temporal Changes Of mir-34 Family TaqMan individual PCR array 16 16 8 * * 8 * * 4 mmu-miR-34a mmu-miR-34a * 4 * 2 * * 2 1 1 01 3 7 15 30 120 01 3 7 15 30 120 8 16 8 * * * 4 2 * mmu-miR-34b-5p mmu-miR-34b-5p * 4 * 2 * 1 01 3 7 15 30 120 1 Fold changes 01 3 7 15 30 120 16 8 mmu-miR-34c 8 4 ** mmu-miR-34c 4 * * 2 * 2 1 01 3 7 15 30 120 1 BMC Genomics 2010, 11:609 For Internal Use Only 01 3 7 15 30 120 Days after ENU treatment - 37 - Sample & Assay Technologies
  38. 38. Top 10 Biological Functions Implicated By Differentially Expressed miRNA Differentially expressed miRNA at day 7 and 15 Target prediction algorithm Top 5% of target genes Ingenuity Pathway analysis BMC Genomics 2010, 11:609 For Internal Use Only - 38 - Sample & Assay Technologies
  39. 39. Summary 2 We introduced the concept of miRNA and miRNA profiling using miScript PCR Arrays. . Exposure to carcinogen (ENU) elicits different miRNA profiles in mouse liver. . miRNA profiles have the potential to serve as biomarkers for genotoxicity. . For Internal Use Only - 39 - Sample & Assay Technologies
  40. 40. Take Home Message Gene expression not only regulates biology, but also serves as a surrogate to monitor biology. . Pathway focused analysis using PCR Array is an easily accessible and accurate platform for biomarker identification and mechanistic studies. . For Internal Use Only - 40 - Sample & Assay Technologies
  41. 41. Pathway-Focused Expression Analysis Complete Experimental Systems: Sample Prep through Data Analysis For Internal Use Only - 41 - Sample & Assay Technologies
  42. 42. Thank You for Attending! Call 1-888-503-3187 to order Email: support@SABioscience.com For Internal Use Only - 42 - Sample & Assay Technologies

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