In an experiment on fruit flies, I have a set of expression data of four groups of larvae: 1. 0 day old mutants 2. 3 day old mutants 3. 5 day old mutants 4. 0 day old wild-type (The wild-type does not have 3 or 5 day olds because by that time wild-type larvae have pupated)
I have a quandary with normalization. Currently, when using R and limma, I normalize only for the comparison I'm doing (e.g. if I was comparing 0 day old mutants to 0 day old wild-types I would only normalize with those two groups and then do the linear modeling / t-tests)
My question is should I normalize across all the groups even if I'm only comparing two groups at a time?
FYI, the assay was performed by Genosensor Corporation of Tempe, AZ using the Genoexplorer microRNA system (http://www.genosensorcorp.com/infoaboutproducts.html) and arrays were scanned using GenePix Pro software version 188.8.131.52. The arrays are single-color (635nm) so I didn't do any within array normalization (as is suggested in limma documentation).