Dear all, I was wondering why there is so many difference between the two following approaches to handle the replication for my experiments. briefly, Here is my target : Cy3 Cy5 wt1 mu1 mu1 wt1 wt2 mu2 mu2 wt2 wt3 mu3 mu3 wt3 to get the gene differentially expressed between Mutant and WT, I have stricly followed the two solutions given in the page 37 of limma userguide (3rd apriol 2010): - the first one (page 37) is using duplicateCorrelation - the second one clearly explicit the design matrix and the contrast matrix (page 38) as follow design = cbind( R1_MuvsWT = c(-1,1,0,0,0,0), R2_MuvsWT = c(0,0,-1,1,0,0), R3_MuvsWT = c(0,0,0,0,-1,1) ) fit = lmFit(MAn,design) cont.matrix = makeContrasts ( MuvsWT = (R1_MuvsWT + R2_MUvsWT+R3_MUvsWT)/3, levels = design ) using these two approaches give quantitatively different results. Which one should I trust ? Thanks by advance for any pieces of advice and / or any help Cheers -- G.M [[alternative HTML version deleted]]