Transcriptome What is it - genome wide transcript abundance How do you obtain it - Arrays + MPSS What do you do with it when you have it - ? Affymetrix GeneChip technology Quality control steps 1. RNA quality 260/280 ratio 2. Array image inspection 3. Hybridization controls 4. % genes present 5. Scaling and normalization factors Affymetrix oligo array Types of Arrays Gene Expression Arrays As name suggests for measuring expression Most genes in Genome covered From a variety of organisms Probe sets are designed to 3 end of gene as labelling of probe starts at end of gene Exon Array Similar to gene expression array Differs in that all exons are covered in probe sets Identifies alternative splicing Tiling arrays Cover Genome sequence For mapping - not for expression Can map a single mutation in a single hydridisation ChiP on Chip analysis ChIP-on-CHIP Identify transcription factor Generate antibody Isolate nuclei Immuno precipitate antibody with isolated genomic DNA Antibody will bind to transcription factor bound to nuclear DNA Isolate and amplify antibody bound DNA Hybridise onto oligo chip Signal where DNA is bound Identify set of genes regulated by a specific transcription factor SNP Chip For detecting Polymorphisms Can detect 50 to 100 thousand on a single chip Can be used to screen for unknown associations duplicated spots in each block 2 x 10k Arabidopsis cDNA array ADNc Hybridation laser 2laser 1 analyse RT Cy3 Cy5 ARN polyA+ Micro-arrays
