Hi I have two questions I used the script below to get pair wise fold change and overall adj P val. library(affy) library(limma) data <- ReadAffy() eset <- rma(data) design <- model.matrix(~ 0+factor(c(1,2,3,4,5,6,1,2,3,4,5,6,1,2,3,4,5,6))) colnames(design) <- c("ni_4dpi", "mock_4dpi", "ormv_4dpi","ni_5dpi", "mock_5dpi", "ormv_5dpi") fit <- lmFit(eset, design) contrast.matrix <- makeContrasts(,levels=design) fit2 <- contrasts.fit(fit, contrast.matrix) fit2 <- eBayes(fit2) tab_all<-topTable(fit2, adjust="BH",number=22810) head(tab_all) This gave me 9 pairs of comparisons with adjusted P values I am trying to repeat it but get the error > contrast.matrix <- makeContrasts(,levels=design) Error in is.character(ej) : 'ej' is missing why? Secondly I am more interested in ormv_4dpi-ni_4dpi and ormv_5dpi -ni_5dpi but, If I specify the 2 pairs to be compared the P values is very bad , P value decreases with the number of pairs in contrast matrix--how? Thanks in advance. Konika