I run a heatDiagram and I am with some doubts. First, when I adjust primary=1 in the function heatDiagram, some of the shown genes are different from those shown by the function topTable when I adjust the coef=1. The opposite also happens. (I considered the same amount of genes in both cases). Why? Second, when I adjust primary=2 and I execute the function heatDiagram, the genes of the other columns cannot be significant. Correct? Please, find below the script that I used and information on the design of the experiment, case this is necessary to help me. Experiment Design Time 1day 2day 3day Rep1 Rep1 Rep1 UnTreated Rep2 Rep2 Rep2 Rep3 Rep3 Rep3 Rep1 Rep1 Rep1 Treated Rep2 Rep2 Rep2 Rep3 Rep3 Rep3 2 treatment (treated and untreated); 3 repetitions, and 3 times. My script (step-by-step) >library(limma) >files <- dir(pattern="\.tol$") >RGtol <- read.maimages(files, columns=list +(Rf="DataVOL",Gf="CtrlVOL",Rb="DataBkgd",Gb="CtrlBkgd")) >printer <- list(ngrid.r=4, ngrid.c=5, nspot.r=16, nspot.c=24, +ndups=2, spacing=1, npins=20, start="topleft") >layout <- list(ngrid.r=4, ngrid.c=5, nspot.r=16, nspot.c=24) >genes.names <- read.table("genes_names.txt", header = TRUE, +sep ="\t", colClasses = "character") >genenames <- uniquegenelist(genes.names[,"Name"],ndups=2,spacing=1) >as.data.frame(genenames) >MAtol <- normalizeWithinArrays(RGtol, layout) >MAtol2 <- normalizeBetweenArrays(MAtol,method="scale") >design <- model.matrix(~ -1+factor(c(1,1,1,2,2,2,3,3,3))) >colnames(design) <- c("time1","time2","time3") >cor.tol <- dupcor.series(MAtol2$M,design,ndups=2,spacing=1,initial=0.7, +trim=0.15,weights=NULL) >fit.tol <- lmFit(MAtol2,design, ndups=2,spacing=1, +correlation=cor.tol$cor,weights=NULL,method="ls") >contrast.matrix <- makeContrasts(time2-time1, time3-time2,time3-time1, +levels=design) >fit2.tol <- contrasts.fit(fit.tol,contrast.matrix) >eb.tol <- eBayes(fit2.tol) >time2.time1tol <- topTable(eb.tol, coef=1, number=50, +genelist=genenames,adjust="fdr") >time3.time2tol <- topTable(eb.tol, coef=2, number=50, +genelist=genenames,adjust="fdr") >time3.time1tol <- topTable(eb.tol, coef=3, number=50, +genelist=genenames,adjust="fdr") >heatdiagram(abs(eb.tol$t),fit2.tol$coef,primary=1,names=genenames, +treatments=colnames(eb.tol$t),orientation="portrait") >heatdiagram(abs(eb.tol$t),fit2.tol$coef,primary=2,names=genenames, +treatments=colnames(eb.tol$t),orientation="portrait") >heatdiagram(abs(eb.tol$t),fit2.tol$coef,primary=3,names=genenames, +treatments=colnames(eb.tol$t),orientation="portrait") >clas.tol <- classifyTests(eb.tol) >vennDiagram(clas.tol) Thanks -- Marcelo Luiz de Laia, M.Sc. Dep. de Tecnologia, Lab. Bioqu?mica e de Biologia Molecular Universidade Estadual Paulista - UNESP Via de Acesso Prof. Paulo Donato Castelane, Km 05 14.884-900 - Jaboticabal, SP, Brazil PhoneFax: 16 3209-2675/2676/2677 R. 202/208/203 (trab.) HomePhone: 16 3203 2328 - www.lbm.fcav.unesp.br - mlaia@yahoo.com