The 1
Affymetrix platform for gene expression analysis Affymetrix recommended QA procedures The RMA model for probe intensity data Application of the fitted RMA model to quality assessment
2
3
Probes are 25-mers selected from a target mRNA sequence. 5-50K target fragments are interrogated by probe sets of 11-20 probes. Affymetrix uses PM and MM probes 4
GeneChip Probe Array
Hybridized Probe Cell
Single stranded, labeled RNA target Oligonucleotide probe
*
*
*
* *
18m
1.28cm
106-107 copies of a specific oligonucleotide probe per feature
>450,000 different probes
Image of Hybridized Probe Array
5 Compliments of D. Gerhold
RNA samples are prepared, labeled, hybridized with arrays, arrrays are scanned and the resulting image analyzed to produce an intensity value for each probe cell (>100 processing steps) Probe cells come in (PM, MM) pairs, 11-20 per probe set representing each target fragment (550K) Of interest is to analyze probe cell intensities to answer questions about the sources of RNA detection of mRNA, differential expression assessment, gene expression measurement
6
7
Look at gel patterns and RNA quantification to determine hybe mix quality. QA at this stage is typically meant to preempt putting poor quality R...
QC.2003.10.09