I am VERY new to R/Bioconductor and over the past two weeks have managed, I hope, to put together a limma script to process my single color generic arrays. I was hoping that some of the experts that read these posts could comment on it - specifically, is it valid? Is there something I could be doing that is easier/more appropriate? I know it works as I have been getting the genelists back out at the end, so, my question is very general...is this how you would process a home-made single color array? If not, I would LOVE some pointers. >library(limma) >targets <- readTargets("targets.txt") >RG <- read.maimages(targets,source="generic" +columns=list(R="Median" ,G="Median",Rb="MedBackground",Gb="MedBackground")) >RG$genes <- read.delim("Annotation file.txt") >BsubRG <- backgroundCorrect(RG, method="normexp", offset=50) >NormRG <- normalizeBetweenArrays(BSubRG$G, method="quantile") >MA <- log2(NormRG) ---Now - if I have a single factor I would set up my design as such: >design <- model.matrix(~0+factor(c(1,2,3,4,1,2,3,4,1,2,3,4))) >colnames(design) <- c("A", "B", "C", "D") >corfit <- duplicateCorrelation(MA, design, ndups=4) >fit <- lmFit(MA, design, ndups=4, correlation=corfit$consensus) >fit <- eBayes(fit) >cont.matrix <- makeContrasts(AvsB=A-B, levels=design) >fit2 <- contrasts.fit(fit, cont.matrix) >fit2 <- eBayes(fit2) >GeneList <- topTable(fit2, "AvsB", n=20, adjust="BH", lfc=1) >write.table(GeneList, file = "file name.txt", quote = FALSE, sep = "\t") ---however, if I have multiple factors, then I set up the design as... >TS <- paste(targets$columnname, targets$columnname, sep=".") >TS <- factor(TS, levels=c("xx.xx", "xx.xx", "xx.xx")) >design <- model.matrix(~0+TS) >colnames(design) <- levels(TS) Everything after is the same as above...in pasting this in I do have a couple of questions. 1. Is it recommended/necessary to apply a Bayesian smoothing to both fits? 2. How do I get back out my gene names *in* the topTable? Thanks for your time and I apologize if the answers seem remarkably obvious. Lisa Lisa Orfe Bustad 405 509-335-6320 "Science is a wonderful thing when one does not have to earn one's living at it." Albert Einstein