Transcript MGG330 L1-2007

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 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
Signal where DNA
is bound
Hybridise onto
oligo chip
Immuno precipitate antibody
with isolated genomic DNA
Antibody will bind to
transcription factor bound
to nuclear DNA
Isolate and amplify antibody bound DNA
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
ADNc
ARN polyA+
laser 1
laser 2
RT
Cy3 Cy5
Hybridation
analyse
http://mpss.udel.edu/tutorial/flash/mpss.swf