Biomarker for genotoxicity 2013

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

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


-2-


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


Clinical Development
Phase III

-3-

Marketed
Drug

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

-4-


Current Hot Spots in Toxicity Risk Assessment

Hepato
Toxicity
Pulmonary
Toxicity

Neuro
Toxicity

Geno
Toxicity

Cardio
Toxicity
Renal
Toxicity


Immuno
Toxicity

-5-


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.


-6-


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.


-7-


Gene Expression Regulates Biology

All require molecular signaling for action


-8-


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.


-9-


Biological Markers Define Processes

Angiogenesis

Inflammation

P53
MDM2
Cyclins
CDKs

VEGF
bFGF
ANGPT
PDGF

IL8
TLR
IFNγ
TNFβ

Individual
participants

Cell cycle


- 10 -


Complete Biological Story
Built on Pathway / Network Analysis

Angiogenesis

Inflammation

Pathway

Cell cycle


- 11 -


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:


- 12 -


Table of Contents

1.


2.

•
•
•

3.

Experimental Design
Results

•
•
•

Experimental Design
Results


- 13 -


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

- 14 -


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


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.

- 16 -


Uniform Amplification Efficiency

Representative set of 500 out of > 4,000 assays used in PCR Arrays
CONCLUSION: Accurate and reliable ∆∆Ct results are guaranteed.


- 17 -


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.

- 18 -


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.


- 19 -


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**

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.

- 21 -

Genes in Signaling Pathways

Genes with High Relevance


Development of Genotoxic Biomarker
Compound Selection


- 22 -


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.

.


- 23 -


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


*RT2 PreAMP for 1 ng

Upload & Analyze Data

- 24 -


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


Scatter Plot

- 25 -

Clustergram

3-D Histogram


Expression Profiles of 11 Signature Genes


- 26 -


Gene Signatures Define Compound Class


- 27 -


Expression Profiles From Different Modes of Action


- 28 -


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.
.


- 29 -


Table of Contents

1.


2.

•
•
•

3.

Experimental Design
Results

•
•
•

Experimental Design
Results


- 30 -


miRNA Biology


- 31 -


miRNAs as Master Regulators


- 32 -


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

.


- 33 -


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

.

.


- 34 -


Temporal Changes In Differentially Expressed miRNA

BMC Genomics 2010, 11:609

- 35 -


Clustering Of Differentially Expressed miRNAs

mir-34 family


- 36 -


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


01 3

7

15

30

120

Days after ENU treatment
- 37 -


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


- 38 -


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.
.


- 39 -


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.
.


- 40 -


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


- 41 -


Thank You for Attending!

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


- 42 -


Biomarker for genotoxicity 2013

Recommended

Recommended

More Related Content

What's hot

What's hot (19)

Similar to Biomarker for genotoxicity 2013

Similar to Biomarker for genotoxicity 2013 (20)

More from Elsa von Licy

More from Elsa von Licy (20)

Biomarker for genotoxicity 2013