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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.
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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 - ?
Quality control steps 1. RNA quality 260/280 ratio 2. Array image inspection 3. Hybridization controls 4. % genes present 5. Scaling and normalization factors
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 Isolate nuclei Generate antibody Identify set of genes regulated by a specific transcription factor Immuno precipitate antibody with isolated genomic DNA Signal where DNA is bound Antibody will bind to transcription factor bound to nuclear DNA Isolate and amplify antibody bound DNA Hybridise onto oligo chip
SNP Chip For detecting Polymorphisms Can detect 50 to 100 thousand on a single chip Can be used to screen for unknown associations
2 x 10k Arabidopsis cDNA array duplicated spots in each block
Micro-arrays ARN polyA+ ADNc laser 1 laser 2 RT Cy3 Cy5 analyse Hybridation
