Dear all, I have the project with the following design (dual color, with dye swap): Cy5 Cy3 Status RepNum Polarity R1 SI31_vs_R1 CTRL+ R1_SI31 R1_CTRL R 1 + R1 SI31_vs_R1 CTRL- R1_CTRL R1_SI31 R 1 - R2 SI31_vs_R2 CTRL+ R2_SI31 R2_CTRL R 2 + R2 SI31_vs_R2 CTRL- R2_CTRL R2_SI31 R 2 - S1 SI31_vs_S1 CTRL+ S1_SI31 S1_CTRL S 1 + S1 SI31_vs_S1 CTRL- S1_CTRL S1_SI31 S 1 - S3 SI31_vs_S3 CTRL+ S3_SI31 S3_CTRL S 3 + S3 SI31_vs_S3 CTRL- S3_CTRL S3_SI31 S 3 - R3 SI31_vs_R3 CTRL+ R3_SI31 R3_CTRL R 3 + R3 SI31_vs_R3 CTRL- R3_CTRL R3_SI31 R 3 - S2 SI31_vs_S2 CTRL+ S2_SI31 S2_CTRL S 2 + S2 SI31_vs_S2 CTRL- S2_CTRL S2_SI31 S 2 - I want to compare R versus S. The first thing I've done is the following (where X is my processed log2(ratio) matrix): ========================================================== status = factor(target$Status) dye = rep(c(1,-1),times = 6) design = model.matrix(~0+dye+status) colnames(design) = c("Dye","R","S") fit <- lmFit(X, design) cont.matrix <- makeContrasts("R-S",levels=design) fit2 <- contrasts.fit(fit, cont.matrix) fit2 <- eBayes(fit2) res1 = topTable(fit2, adjust="BH", number = Inf, coef="R-S") ========================================================== which raise 93 transcript significantly differentially expressed. Then, I've tried to use the mixed model approach which raise about 4000 transcripts : ========================================================== MAmixed = normalizeBetweenArrays(RG,method = "Aquantile") # replace NA using KNN MnoNa = gestionNA_knn(MAmixed$M) AnoNa = gestionNA_knn(MAmixed$A) MAmixed$M = MnoNa MAmixed$A = AnoNa t2 = targetsA2C(target) u <- unique(t2$Status) f <- factor(t2$Status, levels=u) design <- model.matrix(~0+f) colnames(design) <- u corfit <- intraspotCorrelation(MAmixed, design) fit <- lmscFit(MAmixed, design, correlation=corfit$consensus) cont.matrix <- makeContrasts("R-S",levels=design) fit2 <- contrasts.fit(fit, cont.matrix) fit2 <- eBayes(fit2) res2 = topTable(fit2, adjust="BH", number = Inf) ========================================================== I was wondering which approach better fit the design of this project. I would also be very grateful if you could (briefly) explain why there is such a difference ? Many thanks by advance. -- Guillaume [[alternative HTML version deleted]]