Thanks a lot for all the suggestions. We finally used both Saroj's and Thomas' suggestions and finished the analysis based on the probesets. The results are satisfying. However, now we are facing new challenges. There are some "one gene - multiple probesets" and "one probeset - multiple genes" issues. If the probesets corresponding to the same gene don't show similar expression, how should we deal with it? Thanks a lot again. On Mon, May 25, 2009 at 8:05 AM, Cheng-Yuan Kao<neokao at="" gmail.com=""> wrote: > We have done affy microarrays for wildtype-control treatment, > wildtype-toxin treatment, mutant-control treatment and mutant-toxin > treatment. > > The goal is to find diffferentially expressed genes regulated by toxin > in wildtype and then find out which of these regulation are mutant > dependent. > > > The first goal is typical. So we did R/ bioconductor - SAM and limma. > Both could give us a bunch of DEGs. However, I am lost about getting > the second aim done. > With limma and 2x2 factorial analysis, we could find the DEGs from all > kind of pairs, such as wildtype -Toxin/control (this answers the first > goal) or mutant/wildtype in control treatment (this tells us how the > mutant gene is affecting the basal expression without toxin). > But I don't know how to find the wildtype DEGs which have regulation > depending on the mutant gene. Say one gene is up-regulated 100 folds > by toxin (i.e. toxin treatment/control treatment) in wildtype. Then if > this gene is up-regulated 3 folds (toxin treatment/control treatment) > in mutant, it will be apparently mutant gene dependent (from 100 folds > in wildtype to 3 folds in mutant). However, this gene will be shown as > DEG in the mutant analysis as well since it is more than the 2-fold > cutoff. Then if I only compared the DEGs in different pair of analysis > from limma, I will miss these kind of genes. There must be some > dedicated way to analyze this but I could not find it. Any suggestion > will be appreciated. Thanks a lot. >