biomarker for genotoxicity 2013

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Sample & Assay Technologies -1- For Internal Use Only Sample & Assay Technologies Pathway-Powered PCR Arrays Bill Wang, Ph.D. QIAGEN Discovery & Validation of Biomarkers to Monitor Genotoxicity by Gene Expression Profiling

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Page 1: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 1 -For Internal Use Only Sample & Assay Technologies

Pathway-Powered PCR Arrays

Bill Wang, Ph.D.QIAGEN

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

Page 2: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 2 -For Internal Use Only Sample & Assay Technologies

Table of Contents

1. Introduction to Genotoxicity

2. Development of Gene-Based In Vitro Genotoxic Biomarkers Using PCR Arrays

• Introduction to RT2 Profiler PCR Array• Experimental Design• Results

3. Identifying miRNA-Based In Vivo Biomarkers to Monitor Genotoxicity in Mouse

• Introduction to miScript PCR Array• Experimental Design• Results

Page 3: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 3 -For Internal Use Only Sample & Assay Technologies

Drug Development At A Glance

7 millions compounds screened

1000 hits

12 candidates

6 candidates 1 product

Early Discovery Phase Exploratory DevelopmentPhase I, II

Clinical Development Phase III

0 5 10 15yr

12 to 24 years

Cost: ~ $900 millionMarketed Drug

Idea

Efficacy and Safety

Page 4: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 4 -For Internal Use Only Sample & Assay Technologies

The inability to accurately predict toxicity early in drugdevelopment cost the pharmaceutical industry billio ns and approximately one-third the cost of all drug failur es.

Drug Discovery Today 2007 12:289-91

Why Toxicity Is Concerned

Reasons For Failure During Drug Discovery & Develop ment Process

FAIL IT EARLIER, FAIL IT CHEAPER !

Page 5: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 5 -For Internal Use Only Sample & Assay Technologies

RenalToxicity

GenoToxicity

CardioToxicity

ImmunoToxicity

PulmonaryToxicity

NeuroToxicity

HepatoToxicity

Current Hot Spots in Toxicity Risk Assessment

Page 6: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 6 -For Internal Use Only Sample & Assay Technologies

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.

Page 7: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 7 -For Internal Use Only Sample & Assay Technologies

Current Testing Battery For Genotoxicity

Bacterial mutagenesis (Ames Test)In vitro mammalian mutagenesis In vitro chromosome aberration assayIn vitro micronucleus assay In vivo chromosome stability assay2 year rodent carcinogenicity test.

Accuracy: 38%, with high false positive rate.

High toxic dose (50-80% growth inhibition) used --- low sensitivityDifficulty at prediction of carcinogenicity at low doses to which human subjects are usually exposed.

For non-genotoxic carcinogens: no suitable model av ailable.

Page 8: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 8 -For Internal Use Only Sample & Assay Technologies

Gene Expression Regulates Biology

All require molecular signaling for action

Page 9: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 9 -For Internal Use Only Sample & Assay Technologies

Gene expression profiles should be profoundly diffe rent between genotoxic and non-genotoxic carcinogens.

Gene expression profiles should be able to discrimi nate among compounds having different mechanisms.

The toxicity of unknown compounds can be predicted by comparison of their molecular fingerprints with tho se obtained with compounds of known toxicity.

Gene Expression And Genotoxicity

Page 10: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 10 -For Internal Use Only Sample & Assay Technologies

Biological Markers Define Processes

Cell cycle Angiogenesis

P53MDM2CyclinsCDKs

VEGFbFGF

ANGPTPDGF

IL8TLRIFNγTNFβIn

divi

dual

pa

rtic

ipan

ts

Inflammation

Page 11: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 11 -For Internal Use Only Sample & Assay Technologies

Pat

hway

Cell cycle Angiogenesis Inflammation

Complete Biological StoryBuilt on Pathway / Network Analysis

Page 12: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 12 -For Internal Use Only Sample & Assay Technologies

Q: How to Assess the Expression of Different mRNAs in a Sample involved in a Pathway and Compare it across Multiple Conditions?

A:

Differential Gene Expression Analysis

Page 13: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 13 -For Internal Use Only Sample & Assay Technologies

Table of Contents

1. Introduction to Genotoxicity

2. Development of Gene-Based In Vitro Genotoxic Biomarkers Using PCR Arrays

• Introduction to RT2 Profiler PCR Array• Experimental Design• Results

3. Identifying miRNA-Based In Vivo Biomarkers to Monitor Genotoxicity in Mouse

• Introduction to miScript PCR Array• Experimental Design• Results

Page 14: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 14 -For Internal Use Only Sample & Assay Technologies

Real-Time PCR

• Amplify & simultaneously quantify target DNA

Reverse Transcription Real-Time PCR

• Amplify & simultaneously quantify messenger RNA (mRNA)

Ct Values

• Threshold Cycle

Principles of qRT-PCR in PCR Arrays

Page 15: Biomarker for genotoxicity 2013

Sample & Assay Technologies

• 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

Anatomy of a PCR Array

Page 16: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 16 -For Internal Use Only Sample & Assay Technologies

Exquisite Specificity

Conclusion: Each well in a PCR Array detects a single gene-specific product.

• 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.

Page 17: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 17 -For Internal Use Only Sample & Assay Technologies

Uniform Amplification Efficiency

CONCLUSION: Accurate and reliable ∆∆Ct results are guaranteed.

Representative set of 500 out of > 4,000 assays used in PCR Arrays

Page 18: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 18 -For Internal Use Only Sample & Assay Technologies

Reproducibility Among Different Instruments

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 .

Reproducibility Among Different Users

High Reproducibility

Page 19: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 19 -For Internal Use Only Sample & Assay Technologies

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 o f a Pathway-Focused set of genes.

Page 20: Biomarker for genotoxicity 2013

Sample & Assay Technologies

** Over 150 Pathway- Powered PCR Arrays Available**

Cancer and Apoptosis Cytokines & Inflammation Develop ment & 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 Neur oscience

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 Custom PCR Arrays (H/M/R/Q/D/F/P/B)

cAMP / Calcium Signaling Atherosclerosis 96-Well, 384-Well Plate

p53 Signaling Diabetes 100-Well Disc, 96x96 Chip

PCR Arrays for ALL Biomedical Researchers

Page 21: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 21 -For Internal Use Only Sample & Assay Technologies

.Biologically relevant gene content• Not simply biochemical pathways or kinase

cascades• Published association with the biological or diseas e

pathway gathered from overlapping sources, including:

� Multiple Publicly Accessible Databases� Text Mining Relevant Literature

Genes in Signaling Pathways

Genes with High Relevance

Technically relevant gene content • Use genes that are regulated at the mRNA level• Specific feedback from thought leaders

How Genes on PCR Arrays Are Selected

Human DNA Damage Signaling Pathway RT 2 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.

Page 22: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 22 -For Internal Use Only Sample & Assay Technologies

Development of Genotoxic BiomarkerCompound Selection

Page 23: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 23 -For Internal Use Only Sample & Assay Technologies

Development of Genotoxic BiomarkerStudy 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.

Page 24: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 24 -For Internal Use Only Sample & Assay Technologies

Non-Genotoxic Genotoxic

Upload & Analyze Data15 minutes

RNA Isolation (RNeasy)RNase-Free DNase Treatment

RNA IsolationAutomatedSolutions

QIAxcel TL IIQIAcube

How RT 2 Profiler PCR Arrays Work

Cells & Tissues RNA (25ng- 5ug)*

1st strand cDNA

~45 minutes

gDNA Elimination Step

First Strand cDNA SynthesisArtificial RNA Spike In (RTC)

*RT2 PreAMP for 1 ng

Real-time PCR Detection of 89 Gene-specific Amplification on

RT2 Profiler PCR Array2 hours

+ SYBR Green Master Mix RGQ

Experiment:

Page 25: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 25 -For Internal Use Only Sample & Assay Technologies

• Gene Content for each Pathway is Pre-Loaded• Custom Arrays: Gene List is all you need

• From Raw C t Values to Fold Change Results in Multiple Analysis Formats

Volcano Plot Scatter Plot Clustergram 3-D Histogram

RT2 Profiler ™ PCR Array Data AnalysisFREE Complete & Easy Analysis with Web-/Excel-Based Software

Page 26: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 26 -For Internal Use Only Sample & Assay Technologies

Expression Profiles of 11 Signature Genes

Page 27: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 27 -For Internal Use Only Sample & Assay Technologies

Gene Signatures Define Compound Class

Page 28: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 28 -For Internal Use Only Sample & Assay Technologies

Expression Profiles From Different Modes of Action

Page 29: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 29 -For Internal Use Only Sample & Assay Technologies

Summary 1

.We described the concept of pathway focused gene ex pression analysis using RT 2 Profiler PCR Arrays.

.We identified 11 genes in DNA damage repair and p53 pathways as a classifier for genotoxic and non-genotoxic com pounds.

.Genotoxic compounds with different modes of action elicit distinct gene expression profiles.

Page 30: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 30 -For Internal Use Only Sample & Assay Technologies

Table of Contents

1. Introduction to Genotoxicity

2. Development of Gene-Based In Vitro Genotoxic Biomarkers Using PCR Arrays

• Introduction to RT2 Profiler PCR Array• Experimental Design• Results

3. Identifying miRNA-Based In Vivo Biomarkers to Monitor Genotoxicity in Mouse

• Introduction to miScript PCR Array• Experimental Design• Results

Page 31: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 31 -For Internal Use Only Sample & Assay Technologies

miRNA Biology

Page 32: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 32 -For Internal Use Only Sample & Assay Technologies

miRNAs as Master Regulators

Page 33: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 33 -For Internal Use Only Sample & Assay Technologies

miScript miRNA PCR Array SystemmiRNA Pathway & Whole miRNome Profiling

.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

miRNA Isolation & cDNA Synthesis

Universal Primer to 40 cycle qPCR

Accurate Results Easy Data Analysis

.Complete Workflow Solution for miRNA Expression Ana lysis

Page 34: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 34 -For Internal Use Only Sample & Assay Technologies

.Goal: explore miRNAs as potential biomarkers for ge notoxicity

.Subject: 6-month old female mice

.Compound: N-ethyl-N-nitrosourea (ENU) 120mg/kg body weight

.Time course:

.Sample source: miRNA from liver

.Analysis: miRNA PCR Array for 384 most expressed mi RNAs

Days

1 3 7 15 30 120

1 30

ENU

DMSO

Mice Days

1 3 7 15 30 120

1 30

Days

1 3 7 15 30 120

1 30

ENU

DMSO

Mice

miRNA Profiling After Exposure To Genotoxic Carcino genStudy Design

Page 35: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 35 -For Internal Use Only Sample & Assay Technologies

Temporal Changes In Differentially Expressed miRNA

BMC Genomics 2010, 11:609

Page 36: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 36 -For Internal Use Only Sample & Assay Technologies

Clustering Of Differentially Expressed miRNAs

BMC Genomics 2010, 11:609

mir-34 family

Page 37: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 37 -For Internal Use Only Sample & Assay Technologies

Temporal Changes Of mir-34 Family

01 3 7 15 30 1201

2

4

8

16

** *

*

mmu-miR-34a

01 3 7 15 30 1201

2

4

8

16

*

**

**

mmu-miR-34a

Fol

d ch

ange

s

Days after ENU treatment

PCR array TaqMan individual

01 3 7 15 30 1201

2

4

8

16

*

** *

*

mmu-miR-34b-5p

01 3 7 15 30 1201

2

4

8

* mmu-miR-34b-5p

01 3 7 15 30 1201

2

4

8

16

* **

*

mmu-miR-34c

01 3 7 15 30 1201

2

4

8

*

mmu-miR-34c

01 3 7 15 30 1201

2

4

8

16

** *

*

mmu-miR-34a

01 3 7 15 30 1201

2

4

8

16

** *

*

mmu-miR-34a

01 3 7 15 30 1201

2

4

8

16

*

**

**

mmu-miR-34a

01 3 7 15 30 1201

2

4

8

16

*

**

**

mmu-miR-34a

Fol

d ch

ange

s

Days after ENU treatment

PCR array TaqMan individual

01 3 7 15 30 1201

2

4

8

16

*

** *

*

mmu-miR-34b-5p

01 3 7 15 30 1201

2

4

8

16

*

** *

*

mmu-miR-34b-5p

01 3 7 15 30 1201

2

4

8

* mmu-miR-34b-5p

01 3 7 15 30 1201

2

4

8

* mmu-miR-34b-5p

01 3 7 15 30 1201

2

4

8

16

* **

*

mmu-miR-34c

01 3 7 15 30 1201

2

4

8

16

* **

*

mmu-miR-34c

01 3 7 15 30 1201

2

4

8

*

mmu-miR-34c

01 3 7 15 30 1201

2

4

8

*

mmu-miR-34c

BMC Genomics 2010, 11:609

Page 38: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 38 -For Internal Use Only Sample & Assay Technologies

Top 10 Biological Functions Implicated By Different ially Expressed miRNA

Differentially expressed miRNA at day 7 and 15

Top 5% of target genes

Target prediction algorithm

Ingenuity Pathway analysis

BMC Genomics 2010, 11:609

Page 39: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 39 -For Internal Use Only Sample & Assay Technologies

Summary 2

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

.Exposure to carcinogen (ENU) elicits different miRN A profiles in mouse liver.

.miRNA profiles have the potential to serve as bioma rkers for genotoxicity.

Page 40: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 40 -For Internal Use Only Sample & Assay Technologies

Take Home Message

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

.Pathway focused analysis using PCR Array is an easi ly accessible and accurate platform for biomarker iden tification and mechanistic studies.

Page 41: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 41 -For Internal Use Only Sample & Assay Technologies

Pathway-Focused Expression AnalysisComplete Experimental Systems: Sample Prep through Data Analysis

Page 42: Biomarker for genotoxicity 2013

Sample & Assay Technologies- 42 -For Internal Use Only Sample & Assay Technologies

Call 1-888-503-3187 to order

Email: [email protected]

Thank You for Attending!