Dear all, I have been reading the previous posts regarding design and contrast matrix, but I am still very confused. I would really appreciate your help as I really need to get my data analysis done ASAP. I have a time course study with six differernt time points and two different genotypes. It was a loop design with dye swap. Here I will only use a four sample smaller loop design for an example: Name FileName Cy3 Cy5 s45 File1 ste4hr ste5hr s4w4 File2 ste4hr wt4hr s54 File3 ste5hr ste4hr s5w5 File4 ste5hr wt5hr w45 File5 wt4hr wt5hr w4s4.1 File6 wt4hr ste4hr w4s4.2 File7 wt4hr ste4hr w54 File8 wt5hr wt4hr w5s5 File9 wt5hr ste5hr I used: > design <- modelMatrix(targets, ref="wt4hr") > design        ste4hr ste5hr wt5hr s45        -1      1     0 s4w4       -1      0     0 s54         1     -1     0 s5w5        0     -1     1 w45         0      0     1 w4s4.1      1      0     0 w4s4.2      1      0     0 w54         0      0    -1 w5s5        0      1    -1 > fit <- lmFit(MA, design) > contrast.matrix <- makeContrasts(wt5hr, ste4hr, wt5hr-ste5hr,ste5hr- ste4hr, levels=design) > fit2 <- contrasts.fit(fit, contrast.matrix) > fit2 <- eBayes(fit2) > results <- decideTests(fit2) My questions: 1. Have I done anything wrong so far? 2. How can I print out a Toptable with gene lists for each contrast? I used the following command, but I think something was wrong because the genes that were identified differentially expressed do not seem to match genepix images at all: Resultfile <- topTable(fit2, coef=1,n=8000,adjust="fdr") (for the first contrast "wt5hr-wt4hr"?) > write.table(Resultfile,"w5w4_8000.txt",sep="\t") 3 Does the number of coef correspond to the order of the contrasts listed in the contrast.matrix command? for example, coef=3 corresponds to the contrast "wt5hr-ste5hr"? I am very sorry for the long questions. I am totally not sure about this contrast matrix thing, and THANK YOU SO MUCH for your help. Thanks -Jenny [[alternative HTML version deleted]]