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
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Sample & Assay Technologies
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
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Sample & Assay Technologies
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
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Clinical Development
Phase III
-3-
Marketed
Drug
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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
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5. Current Hot Spots in Toxicity Risk Assessment
Hepato
Toxicity
Pulmonary
Toxicity
Neuro
Toxicity
Geno
Toxicity
Cardio
Toxicity
Renal
Toxicity
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Immuno
Toxicity
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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.
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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.
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8. Gene Expression Regulates Biology
All require molecular signaling for action
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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.
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11. Complete Biological Story
Built on Pathway / Network Analysis
Angiogenesis
Inflammation
Pathway
Cell cycle
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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:
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Sample & Assay Technologies
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. 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
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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
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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.
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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.
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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.
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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.
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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**
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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.
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Genes in Signaling Pathways
Genes with High Relevance
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22. Development of Genotoxic Biomarker
Compound Selection
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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.
.
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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
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*RT2 PreAMP for 1 ng
Upload & Analyze Data
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Sample & Assay Technologies
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
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Scatter Plot
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Clustergram
3-D Histogram
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26. Expression Profiles of 11 Signature Genes
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27. Gene Signatures Define Compound Class
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28. Expression Profiles From Different Modes of Action
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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.
.
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Sample & Assay Technologies
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
32. miRNAs as Master Regulators
For Internal Use Only
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Sample & Assay Technologies
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
.
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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
.
.
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35. Temporal Changes In Differentially Expressed miRNA
BMC Genomics 2010, 11:609
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36. Clustering Of Differentially Expressed miRNAs
mir-34 family
BMC Genomics 2010, 11:609
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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
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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.
.
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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.
.
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