Transcript Microarray production

Microarrays overview,
one and two colour systems
Richard Talbot
Roslin Institute and R(D)SVS
University of Edinburgh
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Microarray uses
• Definition
• A microarray is a high density of DNA probes of
known sequence arrayed across a solid surface
• Uses
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Gene Expression
Comparative Genome Hybridisation (CGH)
Single Nucleotide polymorphisms (SNP)
DNA methylation
Copy Number variation (CNV)
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Microarray experiment
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Define the questions
Choose microarray
RNA preparation
Target labelling
Array Hybridisation
Image scanning and analysis
Statistical analysis
Reporting results
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Experimental model
Gene expression in the developing limb
model: During development different genes
are expressed in a choreographed order for
the spatial patterning of tissues. Chick limb
buds at stage 20 (~3 days) show patterning of
Sonic Hedgehog a major transcription factor
controlling the anterior-posterior axis in the
limb bud.
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Experimental questions
1. What are the genes associated with
patterning in the limb bud?
2. What are the genes influenced by the
presence of Sonic Hedgehog protein?
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Experimental plans
Question 1
Posterior
expression
V’s
Anterior
expression
Anterior bead
expression
V’s
Posterior
expression
Anterior
expression
V’s
Anterior Bead
expression
Anterior Gli3
expression
V’s
Posterior
expression
Anterior
expression
V’s
Anterior GLi3
expression
Question 2
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Reference design
One Colour Experiment
Reference
Anterior bead
expression
Anterior
expression
Posterior
expression
Anterior Gli3
expression
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Microarray experiment
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Define the questions
Choose microarray
RNA preparation
Target labelling
Array Hybridisation
Image scanning and analysis
Statistical analysis
Reporting results
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Types of Microarrays
• Probe type
• cDNA
• Oligonucleotide short
(<35 bases)
• Oligonucleotide long
(>35 bases)
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Oligonucleotide synthesis
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Microarray experiment
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Define the questions
Choose microarray
RNA preparation
Target labelling
Array Hybridisation
Image scanning and analysis
Statistical analysis
Reporting results
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
RNA quality
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Microarray experiment
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Define the questions
Choose microarray
RNA preparation
Target labelling
Array Hybridisation
Image scanning and analysis
Statistical analysis
Reporting results
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
RNA Labelling
Indirect
Direct
Total RNA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
Cy3*-CTP or Cy5*-CTP
Aminoallyl-UTP
Reverse
transcription
cDNA
a
a a
a
cDNA
a
a a
Dye Coupling
Cy3* or Cy5*
a
a a
a
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a
a
a a
a
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ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
RNA amplification
Total RNA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
Reverse
transcription
cDNA
TTTTTT-T7
AAAAAA-T7
TTTTTT-T7
AAAAAA-T7
TTTTTT-T7
AAAAAA-T7
TTTTTT-T7
AAAAAA-T7
Remove RNA
and ds-cDNA
cRNA
In Vitro
transcription
T7
T7
T7
T7
T7
T7
T7
T7
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Affymetrix Single Channel
Microarray Experiment
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Microarray experiment
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Define the questions
Choose microarray
RNA preparation
Target labelling
Array Hybridisation
Image scanning and analysis
Statistical analysis
Reporting results
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Microarray Experiment
Control RNA
Treatment RNA
Reverse Transcription
Dye Coupling
Cy5
Cy3
Excitation
Mix on
Slide
Emission
Scanning
Hybridise
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Hybridisation
•Temperature control
•Mixing
•Multi-chamber
•Loading sample
•Washing
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Scanning
•Set laser power
•Set PMT gain or auto adjust
•Set scan area
•Apply griding file
•Align griding file
•Flag problem features
•Extract Values
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Microarray experiment
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Define the questions
Choose microarray
RNA preparation
Target labelling
Array Hybridisation
Image scanning and analysis
Statistical analysis
Reporting results
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Sources of variation 1
Microarray production
• Print-pin anomalies
• Variations in printed probe
quantities even with the same
pin
• Chip batch variation (due to
many sources of unknown
variations)
• Differences in sequence length
of the immobilized DNA
• Variations in chemical probe
attachment levels to the slide
mRNA
• Differences in conditions.
• Differences between
experimental subjects within
the same covariate level.
• Differences between samples
from the same subject.
• Variation in mRNA extraction
methods from original sample.
• Variations in reverse
transcription.
• Differences in PCR
amplification.
• Different labelling efficiencies.
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Sources of variation 2
Hybridization process
• Different dye sensitivities.
• Inequalities in the application
of mRNA to the slide.
• Variations in the washing
efficiencies of non-hybridized
mRNA on the slide.
• Other differences in
hybridization parameters,
such as:
Scanning/imaging
• Different scanners.
• Different photo-multiplier
gain.
• Different spot-finding
software.
• Different grid alignments.
• Different laser power
• temperature
• experimenter
• time of the day
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Normalisation (two dye)
• Used to remove systematic
bias form the image data
• Methods
• Global
• All genes
• Housekeeping/spike genes
• Loess
• Global
• Pin tip
• Spatial
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Normalisation (one dye)
Method depends on slide system
• Affymetrix
• MAS5.0
• RMA
• Plier
• Agilent
• Spatial normalisation
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Differential expression
• T-Test
• Multiple testing - False Discovery Rate (FDR)
• ANOVA
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Microarray experiment
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Define the questions
Choose microarray
RNA preparation
Target labelling
Array Hybridisation
Image scanning and analysis
Statistical analysis
Reporting results
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Minimal Information About a Microarray Experiment
• Database of microarray experiment
Protocols
Experiment
Hybridisation
ARRAY
Samples
Data Analysis
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Gene Expression Data Repositories
• Major public repositories
• ArrayExpress (EBI) - http://www.ebi.ac.uk/microarray-as/ae/
• GEO (NCBI) - http://www.ncbi.nlm.nih.gov/geo/
• CIBEX (DDBJ) – http://cibex.nig.ac.jp/index.jsp
• Submission tools
• Tab2Mage - http://www.ebi.ac.uk/cgi-bin/microarray/tab2mage.cgi
• GEOarchive http://www.ncbi.nlm.nih.gov/projects/geo/info/spreadsheet.html#G
EOarchive
• Cibex - http://cibex.nig.ac.jp/submission/index.jsp
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
After the Gene list
Electronic
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Annotation
Clustering
Pathways
Text mining
Networks
Practical
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qPCR
In Situ hybridisation
Northern blot
Western Blot
Immunohistochemistry
siRNA
Pharmacology
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals
Conclusions
• Several successful microarray platforms are available
• Choice of platform is based on experimental design
and cost
• It is key to know the questions being asked
• Many steps in a microarray experiment contribute to
the overall variance
• Normalisation can be used to remove technical
variance
• Microarrays are a tool to answer a specific question
have a plan on how to confirm the answer.
ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals