I have tried to search the BioC archives but have not found posts dealing with both paired samples and technical replicates in a setup like mine. I have 3 patients A, B, and C from which I have 4 samples: M.early, nM.early, M.late, and nM.late. Each sample has a technical replicate resulting in 24 arrays (Affymetrix). biolrep Patient M.early nM.early M.late nM.late 1 A 1 0 0 0 1 A 1 0 0 0 2 A 0 1 0 0 2 A 0 1 0 0 3 A 0 0 1 0 3 A 0 0 1 0 4 A 0 0 0 1 4 A 0 0 0 1 5 B 1 0 0 0 5 B 1 0 0 0 6 B 0 1 0 0 6 B 0 1 0 0 7 B 0 0 1 0 7 B 0 0 1 0 8 B 0 0 0 1 8 B 0 0 0 1 9 C 1 0 0 0 9 C 1 0 0 0 10 C 0 1 0 0 10 C 0 1 0 0 11 C 0 0 1 0 11 C 0 0 1 0 12 C 0 0 0 1 12 C 0 0 0 1 I would like to find the following 4 contrasts: 1) M.early - nM.early 2) M.late - nM.late 3) M.early - M.late 4) nM.early - nM.late biolrep <- c(1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,12,12) corfit <- duplicateCorrelation(eset, design, ndups=1, block=biolrep) If I ignore the pairing part I have constructed the following design matrix: > design M.early nM.early M.late nM.late 1 1 0 0 0 2 1 0 0 0 3 0 1 0 0 4 0 1 0 0 5 0 0 1 0 6 0 0 1 0 7 0 0 0 1 8 0 0 0 1 9 1 0 0 0 10 1 0 0 0 11 0 1 0 0 12 0 1 0 0 13 0 0 1 0 14 0 0 1 0 15 0 0 0 1 16 0 0 0 1 17 1 0 0 0 18 1 0 0 0 19 0 1 0 0 20 0 1 0 0 21 0 0 1 0 22 0 0 1 0 23 0 0 0 1 24 0 0 0 1 attr(,"assign") [1] 1 1 1 1 attr(,"contrasts") attr(,"contrasts")$f [1] "contr.treatment" fit <- lmFit(eset, design, ndups=1, block=biolrep, cor=corfit$consensus) contrasts <- makeContrasts(M.early-nM.early, M.late-nM.late, M.early-M.late, nM.early-nM.late, levels=design) fit2 <- contrasts.fit(fit, contrasts) fit2 <- eBayes(fit2) However, I would like to include the pairing by patient into the analyses and that I cannot figure out how to do. Hope someone can help, Charlotte