Hi, I am very new to bioconductor and microarray analysis in general. I am using Windows XP and R version 2.5.1. I have a few questions about the limma package that I am using for cDNA array analysis. First: I would like to flag out all spots with fluorescence intensity lower than that of the mean background intensity of the array. The weight function I am using to do this is (using Genepix output file (*.gpr) column names): myfun<-function(x){ okred<-mean(x[,"B635"])-x[,"F635 Mean"]<=0 okgreen<-mean(x[,"B532"])-x[,"F532 Mean"]<=0 as.numeric(okgreen & okred) } Does this actually do what I want? I?m not certain of how to calculate ?mean background intensity? for the whole array. Also, when spots are given a weight of zero, are only spots that have weight >0 across all replicate arrays included in the analysis? How do I find the number of spots included in the analysis? I know how to do this for individual arrays but if arrays 1-4 (out of 16) are in group 1, how do I find how many spots are included in the analysis for group 1? Second: Two of my array groups consistently have all significant spots being ?downregulated? relative to the control. I feel that there must be an error in my normalization step or background correction step. This is the code I am using: #Background correction and normalization RG2<-backgroundCorrect(RG, method="normexp", offset=50) w<-modifyWeights(RG2$weights, RG2$genes$Status, c("Actin","Buffer"), c(2,2)) MA<-normalizeWithinArrays(RG2, method="loess", weights=w) MA.Aq<-normalizeBetweenArrays(MA) #look at array weights arrayw<-arrayWeightsMA.Aq) #Create the design matrix for linear models design<-modelMatrix(targets, ref="control") #fit a linear model to each gene fit<-lmFitMA.Aq, design=design, weights=arrayw) #create a contrast matrix to compare each treatment to the control case contrast.matrix<-makeContrasts(Acp26Aa, Acp29Ab, Acp36DE, Acp62F, levels=design) #expand linear model and compute empirical Bayes statistics fit2<-contrasts.fit(fit, contrast.matrix) fit2<-eBayes(fit2) Is normexp the best way to do background correction? Also, this array (DGRC-1) is printed with 48 pins, so I?m pretty sure I can?t use printtiploess. Is this true? Finally, what is the underlying linear model that lmFit is using? I can find no information on this. I assume it treats treatment and dye as fixed effects and array as a random effect, but I just want to confirm this. Thank you so much for any help demystifying this. Sorry this was so long. Brooke L.