[BioC] Affymetrix quality control and normalization
Dipl.-Ing. Johannes Rainer
johannes.rainer at tugraz.at
Tue Jul 27 08:36:08 CEST 2004
we got a new affymetrix station and are now becoming a affymetrix core facility
. as i am fairly new in the one color micro array field i wanted to know how
other people work with affymetrix chips.
at the moment i am normalizing the chips with RMA. i compared these results with
MAS5 and GC-RMA background correction. from this comparsions it looked that RMA
worked best (also with only two chips used in the comparsion), GC-RMA made some
strange adjustements (i found genes down regulated after GC-RMA background
correction, where they should (must) be up regulated). with MAS 5 i get to many
regulated genes, to big variance in the low intensity range... so the method i
am using now is RMA.
now to the questions:
a) quality control: how to define when a chip has not worked, when excluding a
chip from the analyis? i am looking at the moment at the histogram (big signal
range or not?) and at the 3' 5' ratio, but where is the limit for this range?
when was the RNA degraded?
b) RMA with a low number of chips, is this possible? i thins the more chips
(biological replicates) i have the better the model fitting workes.
c) defining regulated genes: i am currently using a M (fold change) cut off of 1
(2 fold), better solutions?
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