Dear list, Just need some place to check my code for the analysis of my array experiment using Limma. As I'm unsure and have a feeling I'm missing something. My experimental setup: 2 groups A and B of 6 samples in a common refrence design all in dye swap using 2 color agilent slides. My targets file looks like this: Name Filename Cy3 Cy5 SampleCy3 SampleCy5 Name FileName Cy3 Cy5 SampleCy3 SampleCy5 A1 A1.txt RF A REF A1 A1ds A1ds.txt A RF A1 REF A2 A2.txt RF A REF A2 A2ds A2ds.txt A RF A2 REF A3 A3.txt RF A REF A3 A3ds A3ds.txt A RF A3 REF A4 A4.txt RF A REF A4 A4ds A4ds.txt A RF A4 REF A5 A5.txt RF A REF A5 A5ds A5ds.txt A RF A5 REF A6 A6.txt RF A REF A6 A6ds A6ds.txt A RF A6 REF B1 B1.txt RF B REF B1 B1ds B1ds.txt B RF B1 REF B2 B2.txt RF B REF B2 B2ds B2ds.txt B RF B2 REF B3 B3.txt RF B REF B3 B3ds B3ds.txt B RF B3 REF B4 B4.txt RF B REF B4 B4ds B4ds.txt B RF B4 REF B5 B5.txt RF B REF B5 B5ds B5ds.txt B RF B5 REF B6 B6.txt RF B REF B5 B6ds B6ds.txt B RF B5 REF Samples: A1-A6 B1-B6 Common refrence: REF RF ds: dyeswap Files contain nomalized R G values for 45220 probes: reporterId R G AA000001 2028.38 3370.22 Loading RG values: data.import.RG <- read.maimages(targets,source="generic",columns=list(R="R",G="G"),other .columns=NULL,annotation=list(ID="reporterId"),verbose=TRUE,sep="\t",q uote="",names=targets$Name) ## apply the design of the experiment design <- modelMatrix(targets, ref="RF") design ## make MA data of the RG data, using normalized data so method=none MAdata <- normalizeBetweenArrays(data.import.RG, method="none") fit <- lmFit(MAdata, design) ## fit contrasts AtoB fitAB <- contrasts.fit(fit,c(1,-1)) fiteB_AB <- eBayes(fitAB) toptable = topTable(fiteB_AB, number=nrow(MAdata), adjust="fdr") write.table(toptable, "toptableAvsB.txt", sep="\t") ## heatmap selected <- p.adjust(fiteB_AB$p.value) <0.05 dim(selected) MA.selected <- MAdata$M[selected, ] dim(MA.selected) hmcol <- maPalette(low="red",high="green",mid="yellow",k=21) heatmap(MA.selected,col = hmcol) So now my questions: 1. is this the right way to do the analysis? I have some feeling that I'm missing something. 2. If I make a heatmap this way I still see dyeswaps, is this ok or am I missing a step? 3. is it possible to make a heatmap of the toptable logFC data? Any pointers are welcome. Regards, Martin