Dear all, I am trying analyze my 2-color microarray data with limma, and I am not sure which design I should use. I have total of 4 arrays:array 1 to 4. array 1 and array 2 are from the same biological experiment with dye swap. array 3 and 4 are from two individualexperiments and also dye swap. I am trying compare gene expression profiles between my mutant 1 (isp-1) and mutant2 (isp-1;ceh-23) For example: Cy3 Cy5array1 isp-1 (exp1) isp-1;ceh-23(exp1)array2 isp-1;ceh-23 (exp1) isp-1 (exp1)array3 isp-1 (exp2) isp-1;ceh-23(exp2)array4 isp-1;ceh-23 (exp3) isp-1 (exp3)I think what I have done is to find the coefficient for each biological experiment and take the average of the coefficients at the end, but I am not sure if this is the best way of doing it.Here's my script : > library(limma) > targets <- readTargets("targets.txt") > RG <- read.maimages(targets,source="agilent.median") Read US83000170_252018610364_S01_GE2_105_Dec08_1_3.txt Read US83000170_252018610364_S01_GE2_105_Dec08_1_4.txt Read US83000170_252018610386_S01_GE2_105_Dec08_1_2.txt Read US83000170_252018610386_S01_GE2_105_Dec08_1_4.txt > RG <- backgroundCorrect(RG, method="minimum", offset=1) > MA <- normalizeWithinArrays(RG, method="loess") > MA.avg <- avereps(MA, ID=MA$genes$SystematicName) > design <- cbind(MU1vsWT1 = c(-1,1,0,0), MU2vsWT2 = c(0,0,-1,0), MU3vsWT3=c(0,0,0,1) ) > fit <- lmFit(MA.avg, design) > cont.matrix <- makeContrasts(MUvsWT = (MU1vsWT1 + MU2vsWT2+MU3vsWT3)/3, levels = design) > fit2 <- contrasts.fit(fit, cont.matrix) > fit2 <- eBayes(fit2) > topTable(fit2, adjust = "BH") > output<- topTable(fit2, adjust = "BH",number=40000) > write.table(output, file="isp-1 vs. isp-1;ceh-23.txt", sep="\t", quote=FALSE) my session info is R version 2.14.0 (2011-10-31)Platform: x86_64-pc- mingw32/x64 (64-bit) locale:[1] LC_COLLATE=English_United States.1252 LC_CTYPE=English_United States.1252 LC_MONETARY=English_United States.1252[4] LC_NUMERIC=C LC_TIME=English_United States.1252 attached base packages:[1] stats graphics grDevices utils datasets methods base other attached packages:[1] limma_3.10.0 I will be appreciate any feedback. thanks a lot! Ella [[alternative HTML version deleted]]