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NASC A Microarray Miscellany David J Craigon NASC 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 NASC I’m going to talk about • • • • • Madison the Conference An interesting poster at Madison Some large-scale microarray projects TAIR Tiling Chips at SALK NASC 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 NASC Madison, WI NASC Madison, WI NASC 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 NASC 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 NASC Microarray information • They used Affymetrix GeneChips • They used the older GeneChips, which represent about 8,200 genes • They extracted RNA from seeds NASC 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 NASC 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 NASC 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 NASC Do Microarrays work? QRT-PCR Microarray QRT-PCR Microarray NASC Next experiment • Microarray time-course experiment over two days • Wild type seed • Simultaneously monitoring levels of gibberellin in the seed with GC-MS. NASC NASC 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. NASC They conclude that GA4 does regulate these genes NASC 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 NASC NASC • 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 NASC And now for something completely different… NASC 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. NASC Advantages • Complete atlas of gene expression possible, with no holes • All experiments can be carried out under compatible conditions • Fewer controls needed NASC AtGenExpress • • • • • • 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 NASC Making the Atlas To be studied: • Developmental stages • Tissue type • Response to light • Response to pathogens • Misc. treatments • Triplicates/ Duplicates NASC 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 NASC NASC NASC Gene Ontology Annotation Progress • TAIR and TIGR perform the annotation • About half the expected genes annotated with GO terms • All from experimental evidence NASC 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 NASC NASC NASC 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 NASC 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. NASC CATMA • Important difference: Based on new genome annotation NASC 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! NASC 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! NASC Signal.salk.edu NASC Signal.salk.edu NASC Signal.salk.edu NASC Signal.salk.edu NASC Signal.salk.edu The latest AGI Annotation NASC I’ve told you about… • • • • • Madison the Conference TAIR An interesting poster Some large-scale microarray projects Tiling Chips at SALK NASC The End! I’ve got references and information about most of what I’ve talked about