Dear Bioconductor, Dear Dr. Smyth, I have hybridizid two color labelled cRNA from five treatments to 10 custom made 8x15k Agilent oligo arrays, in a saterated/unconnected design. I am looking for differentially expressed genes between the five treatments. QC reports from the Agilent Feature Extraction Software are good (e.g. nice observed/expected R^2 around 0.99). For separate channel analysis you need to calculate the consensus correlation of intraspot correlation between red and green intensities. This is in my case very low (0.06922669). On one of the threads from the past I read that this should be 0.8-0.9. What can be the reason for this low correlation coefficents? I am convinced that the function intraSpotCorrelation calculates the R and G values from the M value, so I do not have to do RG.MA(), right? I can not get rid of the intuition that this function calculates the correlation between the M and A values, then my observed consensus correlation would make sense. When I manually set the consensus.correlation to, let's say, 0.9 I do get sets of differentially expressed genes (but of course this is not the way because I want to know what is going on ...!). For the interested reader, my script is below here. I would be very grateful to get some feedback on this topic. Chapter 9 of the limma usersguide, concerning this topic, is rather short. kind regards, Thierry Janssens library(limma) library(statmod) limmaUsersGuide(view=TRUE) setwd("C:/Documents and Settings/thierry/My Documents/Data/Swantje Staaden/BioC") filenames <- readTargets("filelist.txt") filenames <- as.vector(filenames[, 2]) RGlist <- read.maimages(files = filenames, source = "agilent") # ... #remove spike-in probes j <- RGlist$genes$ControlType == 0 #normalize within arrays RGbc <- backgroundCorrect(RGlist, method = "edwards", offset = 30) MA <- normalizeWithinArrays(RGbc[j, ], method ="loess") # MA plotjes pdf("MAplotsnorm.pdf") for(i in 1:10) {plotMA(MA, array = i) abline(h=0, col = "red")} dev.off() #normalize between arrays MAbet <- normalizeBetweenArrays(MA, method = "Aquantile") pdf("densityplots.pdf") plotDensities(MAbet) dev.off() #sort on duplo o <- order(MA$genes$ProbeUID) MAsorted <- MA[o,] o <- order(MAbet$genes$ProbeUID) MAbetsorted <- MAbet[o,] r <- 0 for(q in seq(1, nrow(MAbetsorted), 3)) { r <- as.numeric((identical(MAbetsorted$genes$probeUID[q], MAbetsorted$genes$probeUID[q+1])) && (identical(MAbetsorted$genes$probeUID[q], MAbetsorted$genes$probeUID[q+2])) ) + r } r # r should be 5069 # Separate channel analysis in limma MAbetsortedav <- avedups(MAbetsorted, ndups = 3, spacing =1) targets <- readTargets("filelist.txt") targetstest <- targetsA2C(targets) u <- unique(targetstest$Target) f <- factor(targetstest$Target, levels=u) design1 <- model.matrix(~0+f) colnames(design1) <- u corfit <- intraspotCorrelation(MAbetsortedav, design1) corfit$consensus.correlation > 0.06922669 # ... etc and the script continues ... > targetstest channel.col Archive Filename Target 1.1 1 File SSArray1.txt B 1.2 2 File SSArray1.txt A 2.1 1 File SSArray2.txt C 2.2 2 File SSArray2.txt B 3.1 1 File SSArray3.txt AC 3.2 2 File SSArray3.txt C 4.1 1 File SSArray4.txt AB 4.2 2 File SSArray4.txt AC 5.1 1 File SSArray5.txt A 5.2 2 File SSArray5.txt AB 6.1 1 File SSArray6.txt C 6.2 2 File SSArray6.txt A 7.1 1 File SSArray7.txt AB 7.2 2 File SSArray7.txt C 8.1 1 File SSArray8.txt B 8.2 2 File SSArray8.txt AB 9.1 1 File SSArray9.txt AC 9.2 2 File SSArray9.txt B 10.1 1 File SSArray10.txt A 10.2 2 File SSArray10.txt AC > design1 B A C AC AB 1 1 0 0 0 0 2 0 1 0 0 0 3 0 0 1 0 0 4 1 0 0 0 0 5 0 0 0 1 0 6 0 0 1 0 0 7 0 0 0 0 1 8 0 0 0 1 0 9 0 1 0 0 0 10 0 0 0 0 1 11 0 0 1 0 0 12 0 1 0 0 0 13 0 0 0 0 1 14 0 0 1 0 0 15 1 0 0 0 0 16 0 0 0 0 1 17 0 0 0 1 0 18 1 0 0 0 0 19 0 1 0 0 0 20 0 0 0 1 0 attr(,"assign") [1] 1 1 1 1 1 attr(,"contrasts") attr(,"contrasts")$f [1] "contr.treatment" -- Thierry K.S. Janssens Vrije Universiteit Amsterdam Faculty of Earth and Life Sciences Institute of Ecological Science Department of Animal Ecology, De Boelelaan 1085 1081 HV AMSTERDAM, The Netherlands Phone: +31 (0)20-5989147 Fax: +31 (0)20-5987123 thierry.janssens at ecology.falw.vu.nl