Hi, Here is my code (thanks to all those who helped me with getting the columns right). library(limma) targets <- readTargets('targets.txt') f <- function(x) as.numeric(x$Flags > -99) RG<-read.maimages(targets$FileName,source="genepix",columns=list(Gf="F 532 Median",Gb="B532 Median",Rf="F635 Media n",Rb="B635 Median")) RG$printer <- getLayout(RG$genes) MA <- normalizeWithinArrays(RG) design <- modelMatrix(targets,ref="Reference") fit<-lmFit(MA,design) fit<-eBayes(fit) After the last line I get this warning: Warning message: Estimation of var.prior failed - set to default value in: ebayes(fit = fit, proportion = proportion, stdev.coef.lim = stdev.coef.lim) To make it even more unnerving is that when I typed "fit" to see what is in there, all the Ref values are NA (in the targets.txt file, I assigned Exp and Ref to various channels). Should I be worried about these NULL values and the warning message? Thanks again! G