ark-genomics: centre for comparative and functional genomics in farm animals richard talbot roslin...

ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals

Richard TalbotRoslin Institute and R(D)SVS

University of Edinburgh

Microarrays overview, one and two colour systems

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• 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

• 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 modelGene 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

Posterior expression

Anterior expression

Question 1

V’s

Anterior bead expression

Posterior expression

V’s

Anterior expression

Anterior Bead expression

V’s

Question 2

Anterior Gli3 expression

Posterior expression

V’s

Anterior expression

Anterior GLi3 expression

V’s

ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals

Reference design

Reference

Anterior bead expression

Anterior expression

Posterior expression

Anterior Gli3 expression

One Colour Experiment

ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals

Microarray experiment

• 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

• 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

• 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 LabellingTotal RNA

cDNA

Reverse transcription

AAAAAAAAAAAA

AAAAAAAAAAAA

Aminoallyl-UTP

a a a a

a a a a

a a a a

a a a a

cDNA

* * * *

* * * *

* * * *

* * * *

Cy3*-CTP or Cy5*-CTP

* * * *

* * * *

* * * ** * * *

Dye CouplingCy3* or Cy5*

Indirect Direct

ARK-Genomics: Centre for Comparative and Functional Genomics in Farm Animals

RNA amplification

Total RNA cDNAReverse

transcription

In Vitrotranscription

Remove RNA and ds-cDNA

AAAAAAAAAAAA

AAAAAAAAAAAA

AAAAAA-T7TTTTTT-T7

AAAAAA-T7TTTTTT-T7

AAAAAA-T7TTTTTT-T7

AAAAAA-T7TTTTTT-T7

T7T7

T7T7

T7T7

T7T7

cRNA

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

• 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

Reverse Transcription

Dye Coupling

Cy3 Cy5

Mix on Slide

Excitation Emission

Hybridise

Scanning

Control RNA Treatment RNA

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

• 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 1Microarray 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

Scanning/imaging• Different scanners.• Different photo-multiplier

gain.• Different spot-finding

software.• Different grid alignments.• Different laser power

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:• 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

• 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

Samples

Data Analysis

Protocols Experiment

ARRAYHybridisation

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#GEOarchive

• 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• Annotation• Clustering• Pathways• Text mining• Networks

Practical• 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.