Hi, I'm trying to use limma to analyze the following: Originally, there were 6 arrays (3 biological reps, each with a dye-swap hybridization). There was a technical issue with one of the arrays, leaving me with an 'unbalanced' design: SlideNumber Name FileName Cy3 Cy5 1 PI-1_vs_DMSO-1 PI-1_vs_DMSO-1.txt PI DMSO 2 DMSO-2_vs_PI-2 DMSO-2_vs_PI-2.txt DMSO PI 3 PI-2_vs_DMSO-2 PI-2_vs_DMSO-2.txt PI DMSO 4 DMSO-3_vs_PI-3 DMSO-3_vs_PI-3.txt DMSO PI 5 PI-3_vs_DMSO-3 PI-3_vs_DMSO-3.txt PI DMSO I'd like to attempt to include the sample without a technical replicate and would like to know if this this would be an acceptable way to handle this situation? ('pool' is a MAList containing 5 the arrays listed in the above order). design <- cbind(PI1vsDMSO1 = c(-1, 0,0, 0,0), PI2vsDMSO2 = c(0, 1,-1, 0,0), PI3vsDMSO3 = c(0, 0,0, 1,-1)) cont.matrix<-makeContrasts(PIvsDMSO = (PI1vsDMSO1 + PI2vsDMSO2 + PI3vsDMSO3)/3, levels=design) fit<-lmFit(pool, design) fit<-contrasts.fit(fit, cont.matrix) fit<-eBayes(fit) Thanks, Ryan -- Ryan Basom Genomics Resource Fred Hutchinson Cancer Research Center Seattle, WA