Hi, >From limma user guide section 8.1.2 Simple comparisons -> Dye swaps it is clear that for the following experimental design: FileName Cy3 Cy5 File1 wt mu File2 mu wt File3 wt mu File4 mu wt *(four replicates of two groups (wt , mu) of which two replicates in each group is labeled by one color(red) the other two is labeled by another color (green)) one can estimate the Dye effect by defining that effect as the intercept : design <- cbind(DyeEffect=1,MUvsWT=c(1,-1,1,-1)) fit <- lmFit(MA, design) fit <- eBayes(fit) Let's say that one finds that the Dye effect (for all genes) is statisticaly insignificant. Would it be correct than to force the model to go through the intercept by design <- cbind(DyeEffect=-1,MUvsWT=c(1,-1,1,-1)) Will I get a better estimation of the Mu to wt ratio? Thanks, Ron