Dear all, We have some arrays in which a large proportion of the spots are expected to be differentially expressed. I would therefore like to normalize using some control spots that we put on the array. I tried up-weighting these spots, as per an example in the Limma users guide. However, when I perform the normalization it pushes all of the spots to have M values extremely close to zero. Am I doing something wrong? When I look at the MAplot before normalization (even after loess) the control spots have a slightly negative M (-0.5). See my script below. Thanks >>>>>>>>>>>>>>>>>>>>>> library(limma) targets<-readTargets("VCMeOHTargets.txt") RG<-read.maimages(targets$FileName,source='genepix',wt.fun=wtflags(0.1 )) RG$genes<-readGAL("Array2(Gal)2008NonDhc.gal") spottypes<-readSpotTypes("SpotTypes.txt") RG<-backgroundCorrect(RG,method="normexp") RG$genes$Status<-controlStatus(spottypes,RG) w<-modifyWeights(array(1,dim(RG)),RG$genes$Status,c("All","Control"),c (0,2)) MAwi<-normalizeWithinArrays(RG,weights=w) MAwibtw<-normalizeBetweenArrays(MAwi,method='Aquantile') design<-c(1,1,-1,-1,1,-1) fit<-lmFit(MAwibtw,design) fit<-eBayes(fit) VCMeOHTop<-topTable(fit,number=10000) write.table(VCMeOHTop, file="VCMeOHTopnormControlAq.txt",sep="\t") plotMA(fit) ****************************************** Alison S. Waller M.A.Sc. Doctoral Candidate alison.waller@utoronto.ca <mailto:alison.waller@chem-eng.utoronto.ca> 416-978-4222 (lab) Department of Chemical Engineering Wallberg Building 200 College st. Toronto, ON M5S 3E5 [[alternative HTML version deleted]]