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Experimental Validation of Microarray Data
Patrick Tan
MD PhD
Talk Outline
- Purposes of Experimental Validation
- Validation in silico
- Northern Blotting
- PCR Assays (measure DNA/RNA)
- Antibody Assays (measure protein)
- Other molecular assays (CGH, SKY)
- Validation Across Centres and Populations
- Phenotypic Validation
What is Validation?
1. To declare or make legally valid.
2. To mark with an indication of official sanction.
3. To establish the soundness of; corroborate.
- How Robust (“sound”) are your findings?
- Can they be replicated?
- Can they be replicated by another laboratory/centre?
- Are they dependent on the specific experimental design?
Why Validate Your Microarray Data?
- To establish confidence in your scientific claims
- To identify areas for future research (eg specific genes)
- May lead to unexpected findings
- A common requirement of reviewers (!)
Potential Sources of Error in Microarray Data
- Cross-hybridization of probe sequences
- Mistakes in Probe Assignment (particularly cDNA arrays)
- Artefacts caused by sample processing (eg RNA amplication)
- Artefacts caused by array experiments (eg Cy3/Cy5 dye-bias)
What is Required for Validation?
Independent Verification :
- Different experimental technique
- Different researchers/laboratories
- Different biological samples
Challenges of Validating Microarray Data
- Microarrays are High-throughput, Validation techniques are Not
- Different scales of comparison - eg different techniques
for normalizing experimental measurements
- Lack of Correlation between Transcriptosome and Proteome
- Not inexpensive (eg Taqman Probes)
A Typical Validation Scenario
Biological Samples
Array Experiment
Statistical Analysis
Differentially
Regulated Genes
(Candidates)
Selection of
Genes for Validation
Experimental Validation
Which Genes are “Suitable” for Validation?
- Strong Regulation
- Abundant absolute expression
- Reasonably well-characterized (Reagents
may be available)
- Relevant to Further Research (ESTs?)
Validation in silico (Validation by internet)
- Have these genes been previously identified in the literature
and shown to be regulated?
- Usually performed to assess data quality or as a
preliminary analysis
Example : In silico Validation
Comparative Genomics of Burkholderia pseudomallei
• Gram negative bacterium.
• Environmental saphrophyte endemic to SEA
• Potential Biowarfare Agent (USA Category B)
• Causative agent of the disease Melioidosis
Use of Whole-Genome
B. pseudomallei DNA Microarrays
A Discovery Platform for
- Strain Typing Markers
- Patterns of Genetic Variation
- Mechanisms of Virulence
Genomes Compared
• B. pseudomallei
– 18 Regional Isolates
• B. mallei
– 2 isolates
- Causative agent of glanders
- Biowarfare Category B
- Distinct Ecological Niche (Non-rhizospheric)
• B. thailandensis
- 3 isolates
- V. closely related to B. pm but clinically avirulent
Array-Based Comparative Genomic Hybridization
Reference
Strain B pm
K96243
Sample Isolate
e.g. B pm #22
B mallei
B thailandensis
Extract Genomic
DNA
Extract Genomic
DNA
Label with Cy5-dCTP
Label with Cy3-dCTP
BP 76
Competitive
Hybridisation
B. pseudomallei vs B. thialandensis
• A cluster of 21 Genes controls Type I O-PS Production in
B. pseudomallei (Reckseidler et.al. (2001))
• 11 genes from this cluster are deleted from B.
thailandensis by Southern Blot analysis
• Are these deletions also present in the microarray
data?
• Are there additional deletions in this cluster?
B. pseudomallei vs B. thialandensis
Genes deleted
Reckseidler et al
Using Southern blot
gmhA
wcbA
wcbC
wcbD
wcbE
wcbF
wcbH
Genes deleted
Microarray
gmhA
manC
wcbA
wcbB
wcbC
wcbD
wcbE
wcbG
wcbH
wcbI
wcbK
wzm2
wzt2
wcbM
wcbN
wcbO
wcbP
wcbQ
wzm
wzt
Total no. of genes: 11
Total no. of genes: 17
wcbO
• 9 genes found
commonly deleted
in both studies.
• 2 genes – wcbF and
wcbK not
represented in our
microarray.
• Additional 7 genes
found deleted using
microarray.
Advantages of in silico Validation
- Can be performed in a batch manner
- Rapid results
- Availability of multiple public databases (eg Pubmed, SAGE, etc)
- Cheap!
Disadvantages
- Requires prior knowledge
- Requires confidence in data from others
- Findings are not Novel
Validation by Northern Blotting
- Arguably still the ‘gold standard’ for validation
- Well-established in the literature
- Experimental properties are well-known
A Typical Northern Blot
RNA Mixture
RNA
RNA
Adapted from http://oregonstate.edu/instruction/bb492/fignames/Southernblot.jpeg
Example of Northern Blotting
“A Global Profile of Germline Gene Expression in C. elegans”
Reinke et al., 2000. Molecular Cell 6, 605-616
- Used C. elegans DNA microarrays to compare the gene
expression profiles of :
(A) Wild-type worms vs Mutants with no germline (glp-4)
(B) Oocyte-only (fem-1) vs Sperm-only (fem-3gf)
Identification of a Candidate Gene : pgl-1
Properties of pgl-1
- Highly expressed in germline (ie downregulated in glp-4)
- Sex-biased expression : increased expression in oocytes)
(ie upregulated in fem-2 vs fem-3gf)
- Increasing expression in successive larval stages (L1-L4)
- Validation of pgl-1 via Northern Blotting
Comparing pgl-1 expression by Northern and Microarray
Microarray:
WT adult oocytes
no germline sperm
adult
L2
adult
L3
adult
L4
Fold change
9.2
1.9
4.8
2.4
1.8
Fold change
(Northern)
100
2.6
12.5
11.1
1.7
Northern:
Control Gene
Kawasaki et al., 1998
Advantages of Northern Blotting
- Very sensitive
- Blots are reusable
- Technical protocol is relatively simple
- Can detect mRNA splice variants
Disadvantages
- Use of radioactivity (although non-radioactive techniques
are available)
- Laborious if many genes need to be tested
- Assay is time-consuming
Microarray Data vs Northern Blotting
- Good preservation of trends
- Microarray Ratios tend to be ‘compressed’ vs Northern
- Northern Blots may detect subtle regulations missed by array
Validation by PCR Methodologies
- May replace Northern Blotting as a ‘gold standard’
- Rapid developments in the field
- Diverse variations on a common technique :
A) Non-quantitative techniques
B) Quantitative techniques
Basic PCR
QuickTime™ and a
GIF decompressor
are needed to see this picture.
From www.faseb.org/opar/bloodsupply/ pcr.html
Major Variations of PCR
Qualitative
Quantitative
Measurement Type
Presence or
Absence
Relative Abundance
Normalization
Binary
Control Genes
Cost
Standard PCR
Specialized Equipment
And Reagents
Example of Qualitative PCR
- Array-CGH Data Set for Burkholderia pseudomallei
- Identified Genes that are Differentially Present Between :
(A) B. pseudomallei vs B. mallei
(B) B. pseudomallei vs B. thailandensis
(C) Between different isolates of B. pseudomallei
Experimental Validation Using Qualitative PCR
ORFs Deleted in B. mallei
P
P P P P T M M
P P
ORFs Deleted in B. thailandensis
P P
P P P T M M
1.5kb –
1.5kb
P P P T M M
P P
P P P T
M M
–
100bp –
100bp –
3548902
3551702
ORFs Deleted in B. mallei and B. thailandensis
P
P P P
P T M M
P
3534302
3534002
ORFs Deleted Between Bpm Isolates
P P P P T M M
1.5kb
P P P P P
P P P P P
3433202
3542302
–
100bp –
3490802
3528002
B. pseudomallei Strain 576 is an Atypical Strain
B. thailandensis
B. mallei
Group I
Group III
Group II
Gene
Function
Probe
Status in 576
rmlA
rmlC
rmlD
wzm
wzt
wbiA
wbiB
wbiC
wbiD
wbiE
wbiF
wbiG
wbiH
wbiI
Orf1
Orf2
Glucose-1-phosphate thymidyltransferase
dTDP-4-keto-6-deoxy-D-glucose 3,5 epimerase
dTDP-4-keto-L-rhamnose reductase
ABC-2 Transporter
ABC-2 Transporter
LPS-O-antigen Acetylase
UDP-glucose 4-epimerase
glycolsyltransferase
dihydroxypolyprenylbenzoate methyltransferase
UDP-hexose transferase
Rhamnosyltransferase
UDP-glucose-4-epimerase
UP-N-acetyl fucosamine transferase
Epimerase/dehydratase
UP-N-acetylglucosaminyltransferase
UDP-glucose-4-epimerase
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Not deleted
--------------------------Not deleted
Not deleted
Strain 576
K96243
Semi-Quantitative PCR
- Compares relative accumulation of amplified products
during the PCR procedure
- Can be performed using standard reagents
- Aliquots are periodically removed from the PCR reaction
(eg 10, 15, 20, 25 cycles) and analyzed on an
agarose gel
Example of Semi-Quantitative PCR
Comparing the Transcriptional Changes Associated with
Maturation of Dendritic Cells
Immature
Dendritic
Mature
Dendritic
Monocytes
Macrophage
- Used cDNA microarrays to compare the gene
expression profiles of Monocytes, Macrophages,
Immature Dendritic and Mature Dendritic cells
Distinguishing Between Related Tissues
Monocytes
Macrophage
Im Dendritic
Mt Dendritic
Candidate Genes :
TARC (Expressed in Dendritic Cells)
RGS1 (Expressed in Monocytes and mDendritic)
Removed after 20 cycles (usu >30 cycles)
1 - Monocytes
2 - Macrophages
3 - Activated Macrophages
4 - Immature Dendritic Cells
5 - Mature Dendritic Cells
Control Gene
QuickTime™ and a
Photo - JPEG decompressor
are needed to see this picture.
Quantitative PCR (“Real-time PCR”)
- Makes use of flouresent labels to monitor progress of
PCR amplification
- Two major methods :
1) Double-stranded DNA binding agents (eg SYBR Green)
2) Sequence Specific Probes (eg Taqman, Mol Beacons)
QuickTime™ and a
GIF decompressor
are needed to see this picture.
From http://www.sigmaaldrich.com/img/assets/6600/sg_ls_mb_pcrxdiagram.gif
Taqman PCR (Exploitation of FRET)
Flouresence from
Reporter is Quenched
Cleavage and Release
Of Reporter
From www.probes.com/handbook/ figures/0710.html
Some Considerations in Quantitative PCR
- Design of Gene-specific Primers
A) Confirm Suitability of Primers for PCR (eg Primer3)
B) Specificity of primer sequences (eg BLAST)
C) 3’ bias of primers
- Selection of Control Genes
A) ‘Housekeeping’ Genes - not expected to vary
eg b-actin, GADPH, 16S Ribosomal RNA
B) Control primers should bind at comparable
efficiency to target sequences
C) Expression of control genes should be comparable
to target sequences
Example of Quantitative PCR
Gene Expression Differencs Between Estrogen Receptor
Postive (ER+ve) And ER Negative Breast Cancers
- Used oligonucleotide microarrays to compare the gene
expression profiles of ER+ve and ER-ve primary
human breast tumors
- ER status predetermined using conventional histopathology
Quantification Graph of ESR1 Gene (3 ER +ve and 3 ER -ve samples)
Calibrator
ERER+
ER+
ERERER+
ER +ve
samples
ER -ve
samples
Melt Curves of ESR1 Gene and 18S rRNA (Control) Gene
ER+ and ERERER+
ER+
ER-
{
{
{
{
ER- {
ER+ {
18S
Results for ESR1 gene
(ER-)
(ER+)
(ER+)
(ER-)
(ER-)
(ER+)
Advantages of Validation by PCR
- Well-accepted standard for validation
- Rapid (approx 30-45 min)
- Requires very small amounts of sample
- Multiple genes can be tested in individual reactions
- Can potentially be multiplexed
Disadvantages
- Need to be careful in primer design (esp 20-mers)
- Quantitative PCR requires special equipment
- Lack of comparisons between different real-time technologies
Break and Questions