Dear Guillaume, The first approach (using duplicateCorrelation) is correct. The second approach accounts for technical variability, but fails to fully account for biological variability of the wt-mu contrast in the standard errors of the tests. The second approach therefore tends to be anti- conservative, and will usually lead to more apparent differential expression than the first approach. The second approach is included in the limma User's Guide only to give users an alternative when the design is too complex for duplicateCorrelation to be applicable. I am assuming that your experiment is made up of dye-swap technical replicates of 3 biological replicates. Best wishes Gordon --------------------------------------------- Professor Gordon K Smyth, NHMRC Senior Research Fellow, Bioinformatics Division, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Vic 3052, Australia. smyth at wehi.edu.au http://www.wehi.edu.au http://www.statsci.org/smyth > Date: Thu, 17 Feb 2011 17:53:04 +0100 > From: Guillaume Meurice <guillaume.meurice at="" igr.fr=""> > To: bioconductor at r-project.org > Subject: [BioC] duplicateCorrelation > > 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 ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:4}}