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 - ?

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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 ArraysGene Expression Arrays

As name suggests for measuring expressionMost genes in Genome coveredFrom a variety of organisms

Probe sets are designed to 3’ end of geneas labelling of probe starts at “end” of gene

Exon ArraySimilar to gene expression arrayDiffers in that all exons are covered inprobe sets

Identifies alternative splicing

Tiling arraysCover Genome sequenceFor mapping - not for expressionCan map a single mutation in a single hydridisationChiP on Chip analysis

ChIP-on-CHIPIdentify 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 DNAHybridise onto oligo chip

Signal where DNA is bound

Identify set of genes regulated by a specific transcription factor

SNP ChipFor detecting PolymorphismsCan detect 50 to 100 thousand on a single chipCan 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

http://mpss.udel.edu/tutorial/flash/mpss.swf