Dear List, I am working with six 27K human cDNA chips.Three of them are for Average Primary Tumor Cells and another three are for Invasive cells.In all chips, the reference in on the red channel, and the tumor samples are on the green channel. Here is my target file: ID FileName Cy3 Cy5 131 A19.gpr APTC Ref 1015 I24.gpr INV Ref 416 A25.gpr APTC Ref 611 A26.gpr APTC Ref 1113 I27.gpr INV Ref 10002 I28.gpr INV Ref We would like to find ratios of gene expression of INVasive vs. APTC.I strictly follow the limma user guide commands in my analysis but I got a problem with lmFit.Here are my R codes: > Group<-factor(targets$Cy3,levels=c("APTC","INV")) > design<- model.matrix(~0+Group) > colnames(design)<-c("APTC","INV") > design APTC INV 1 1 0 2 0 1 3 1 0 4 1 0 5 0 1 6 0 1 attr(,"assign") [1] 1 1 attr(,"contrasts") attr(,"contrasts")$Group [1] "contr.treatment" > SNR<- function(x,threshold=1){ + okred<-x[,"SNR 635"]>=threshold + okgreen<-x[,"SNR 532"]>=threshold + okflag<- x[,"Flags"]==0 + as.numeric(okgreen & okred & okflag) + } > RG<-read.maimages(targets$FileName,source="genepix",wt.fun=SNR) Read A19.gpr Read I24.gpr Read A25.gpr Read A26.gpr Read I27.gpr Read I28.gpr > RG<-backgroundCorrect(RG,method="normexp",offset=50) Green channel Corrected array 1 Corrected array 2 Corrected array 3 Corrected array 4 Corrected array 5 Corrected array 6 Red channel Corrected array 1 Corrected array 2 Corrected array 3 Corrected array 4 Corrected array 5 Corrected array 6 > MA<-normalizeWithinArrays(RG,weights=RG$weights) > fit<-lmFit(MA,design) Warning message: In lmFit(MA2, design) : Some coefficients not estimable: coefficient interpretation may vary. I don't really understand this warning message. Is there anything wrong with my design matrix?Thank you in advance for any suggestions or comments! Best Regards, Cathy Albert Einstein Cancer Center