Dear Bioconductor: I have a question concerning normalization of data from Affymetrix Drosophila chip .cel files. This is a general question about how to normalize over multiple treatments. I have data from multiple (4) treatments with 3-6 biologically independent arrays for each treatment. I also have 5 biologically independent control arrays -- the same 5 arrays serve as controls for comparison to each of the treatments. I have used RMA to compare each treatment separately against the controls to determine genes whose expression levels change significantly under each treatment. As a result, I normalized each of the four treatments to the controls separately -- that is I did four separate normalizations with the one set of controls and one set of treatment arrays. Would it be more appropriate to normalize all of the the experimental arrays in the analysis to the controls at once rather than separately, one treatment at a time (as described above)? Many thanks. Paul Mack, Ph.D Department of Genetics University of Georgia Athens, GA USA 706-542-1578 (w) 706-542-3910 (fax) paulmack@arches.uga.edu