Dear List, I am using limma to analyze a gene expression microarray experiment, similar to the one described in the Monograph (Chapter 23.5: Technical Replication, page 404): a knock-out versus wt (BIO effect) 2-color mouse experiment, using dye-swaps (DYE effect) and two independent biological replicates (GROUP effect). My problem is that I get a lot of differences of unexpectedly high significance, even for expression changes as low as 1.4-fold (e.g. log2ratio=0.48, fdr=0.015, see below). My questions are: Is the design correct, as the result of the fitted model has zero degrees of freedom? If so, can you comment the high significance of subtle expression changes? If not, - should I choose which effect (DYE or GROUP) to include in the model, along with the BIO one, based on their significance? - is the fitting itself considered a bug? Details and code SlideNumber Cy3 Cy5 1 wt1 ko1 2 ko1 wt1 3 wt2 ko2 4 ko2 wt2 # After normalizing the data using: RG1none <- backgroundCorrect(RG1, method="none") MARG1none <- normalizeWithinArrays(RG1none, method= "printtiploess") MARG1none <- normalizeBetweenArrays(MARG1none, method= "Aquantile") # using the design: design <- cbind (ko1vswt1= c(1, -1, 0, 0), ko2vswt2= c(0, 0, 1, -1), dye= c(1, 1, 1, 1)) # subsetting the non-control reporters MouseGenes <- MARG1none$genes$Status== "Mouse" # fitting the models fit <- lmFit(MARG1none[MouseGenes, ], design) cont.matrixBIO <- makeContrasts (KOvsWT= (ko1vswt1 + ko2vswt2)/2, levels= design) cont.matrixGROUP <- makeContrasts (Group1vsGroup2= (ko1vswt1 - ko2vswt2), levels= design) cont.matrixDYE <- makeContrasts (DYE= dye, levels= design) fitBIO <- contrasts.fit (fit, cont.matrixBIO) fitGROUP <- contrasts.fit (fit, cont.matrixGROUP) fitDYE <- contrasts.fit (fit, cont.matrixDYE) fitBIO <- eBayes (fitBIO) fitGROUP <- eBayes (fitGROUP) fitDYE <- eBayes (fitDYE) >topTable(fitBIO, num=5000, adjust="fdr") ^^^^^^^^ Block Row Column ID Name Status logFC AveExpr .. 19095 29 6 14 MMAA300016782 MMAA300016782 Mouse 0.4823145 12.392166 t P.Value adj.P.Val B .. 19095 3.930997 2.383282e-03 1.523999e-02 -2.862820232 > R.version # Win XP SP2 _ platform i386-pc-mingw32 .. version.string R version 2.4.1 Patched (2007-01-10 r40440) > .Platform$GUI # GNU Emacs 21.3.1, ESS version 5.3.3 [1] "RTerm" > package.version("limma") [1] "2.9.11" Thank you all and especially Dr Smyth for their wonderful work. Best regards, Thanasis PS I am a biologist and this is my first post to the list. Thanasis Margaritis Institute of Molecular Biology and Biotechnology FORTH, Vassilika Vouton P.O.Box 1385 GR 711 10 Heraklion, Crete GREECE Tel: +30 2810 391927