Transcript A MIAME-compliant Microarray Database

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A Microarray Miscellany
David J Craigon
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A case for the Trade
Descriptions Act?
• This talk was billed to be
about “Microarray related
things we saw at Madison”
• In fact, some of the things
here were not seen at
Madison
• And some of the things have
nothing to do with
Microarrays
• And none of things have
really got much in common
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I’m going to talk about
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Madison the Conference
An interesting poster at Madison
Some large-scale microarray projects
TAIR
Tiling Chips at SALK
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The Madison Meeting
• AKA The 14th
International
Conference on
Arabidopsis Research
• Held every year
• Every two years, held in
Madison U.S.A.
• Last year Seville
• Next year Berlin
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Madison, WI
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Madison, WI
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So, what sort of Microarray
work was on show?
• Microarray have become very common
• Lot of posters about microarray work
• Very few posters use microarrays as part of a
wider pattern of research
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RIKEN Poster
• One poster where the authors did more than
just a microarray
• Interested in how Gibberellin stimulates seed
germination.
• I’m going to focus on the technical aspects
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Microarray information
• They used Affymetrix GeneChips
• They used the older GeneChips, which
represent about 8,200 genes
• They extracted RNA from seeds
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Their first experiment
Germination Time
100
90
80
70
% germinated
• A gibberellin deficient
mutant, ga1-3
• Will germinate if
exposed to Gibberellin14
• Does not germinate
under control conditions
60
With Gibberellin
50
Control
40
30
20
10
0
0
12
18
24
Time (hours)
36
48
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Their first experiment
• Time course after exposure to GA14
• Found some up regulated (230) and down
regulated genes (127)
• Chose 30 transcripts to check by QRT-PCR
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Do Microarrays work?
Affymetrix vs QRT-PCR
1.200
R2 = 0.766
Affymetrix
1.000
0.800
0.600
0.400
0.200
0.000
0.000
0.200
0.400
0.600
QT-PCR
0.800
1.000
1.200
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Do Microarrays work?
QRT-PCR
Microarray
QRT-PCR
Microarray
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Next experiment
• Microarray time-course experiment over two
days
• Wild type seed
• Simultaneously monitoring levels of
gibberellin in the seed with GC-MS.
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Some clustering
• In the first, mutant, experiment we found
some GA4 sensitive genes
• Let’s cluster them over our time course in the
second experiment.
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They conclude that GA4 does regulate these genes
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GFP fusions
• In the first experiment, they found some
genes regulated by addition of GA-4
• They made GFP fusions to three of these
genes
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• They go on to do promoter analysis, and talk
about interactions with ethylene and auxin
• I liked this poster/paper because it showed
microarrays used as part of a wider program
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And now for something
completely different…
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International Transcriptomic
Projects
• Many places, like NASC, have been funded to
do run a lot of microarrays
• If you are going to run lots of microarrayswhy not form a coordinated project to use all
of them?
• Creation of coordinated transcriptome
references.
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Advantages
• Complete atlas of gene expression possible,
with no holes
• All experiments can be carried out under
compatible conditions
• Fewer controls needed
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AtGenExpress
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Uses Affymetrix ATH1 chips
Based in Germany, run by Thomas Altmann
Not actually funded yet
1,300 slides
100 slides to be run at NASC
Data to TAIR
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Making the Atlas
To be studied:
• Developmental stages
• Tissue type
• Response to light
• Response to pathogens
• Misc. treatments
• Triplicates/ Duplicates
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TAIR and Microarrays
• TAIR intend to hold Arabidopisis Microarray
data in their database, including
AtGenExpress
• At the moment they have AFGC data, plus a
few others
• Analysis tools available
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Gene Ontology Annotation
Progress
• TAIR and TIGR perform the annotation
• About half the expected genes annotated
with GO terms
• All from experimental evidence
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Classifing genes
• The gene ontology is used to classify genes
• An ontology is a controlled vocabulary.
• Contains information about how words in the
ontology relate to one another
• Allows computers to understand annotation
as well as humans
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CAGE
• The Compendium of Arabidopsis Gene
Expression
• Another project to do broadly the same thing
• Key difference- to be run using CATMA-based
spotted arrays
• Centres all over Europe
• Data to be handled by the EBI
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What’s CATMA then?
• CATMA is a collection of GSTs- amplified
sections of the genome for genes
• Useful for making spotted arrays- complete
genome coverage
• NASC has a set and will distribute
• Not just good for microarrays- large-scale full
genome RNAi lines.
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CATMA
• Important difference: Based on new genome
annotation
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Genome annotation is in a
state of flux
• Have we found all the genes?
• Are all the genes that we predict really there?
• Have we got the gene models right?
• Can microarrays help us answer these
questions? Of course!
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Tiling chips
Principle:
• Design a series of Affymetrix chips that
covers the entire genome!
• Run RNA extracts from plants under a wide
range of conditions on these chips.
• See which areas of the genome light up!
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Signal.salk.edu
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Signal.salk.edu
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Signal.salk.edu
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Signal.salk.edu
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Signal.salk.edu
The latest AGI Annotation
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I’ve told you about…
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Madison the Conference
TAIR
An interesting poster
Some large-scale microarray projects
Tiling Chips at SALK
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The End!
I’ve got references and information
about most of what I’ve talked
about