Dear All, I have one question that makes me perplex, and no message concerning this problem is described in the mailing list. I'm using the Limma package for the comparison of two experimental conditions (KO vs WT) in a 'reference design' with dye-swap. My experimental design is the following: Cy5 Cy3 Array1 Ref WT Array2 Ref WT Array3 Ref WT Array4 WT Ref Array5 WT Ref Array6 WT Ref Array7 Ref KO Array8 Ref KO Array9 Ref KO Array10 KO Ref Array11 KO Ref Array12 KO Ref In such design it is possible to estimate a dye effect. First, I have fitted a very simple model. > fit1 <- lmFit(MA, design=design1) where design1 is the following design matrix: > design1 KO WT 208_REF-WT.1B 0 -1 213_REF-WT.25B 0 -1 301_REF-WT.3B 0 -1 210_WT.2B-REF 0 1 215_WT.26B-REF 0 1 304_WT.5B-REF 0 1 209_REF-KO.28B -1 0 214_REF-KO.27B -1 0 303_REF-KO.13B -1 0 211_KO.18B-REF 1 0 300_KO.15B-REF 1 0 302_KO.17B-REF 1 0 Then I have fitted a second model including dye effect. > fit2 <- lmFit(MA, design=design2) where design2 is the following design matrix: > design2 DyeEffect KO WT 208_REF-WT.1B 1 0 -1 213_REF-WT.25B 1 0 -1 301_REF-WT.3B 1 0 -1 210_WT.2B-REF 1 0 1 215_WT.26B-REF 1 0 1 304_WT.5B-REF 1 0 1 209_REF-KO.28B 1 -1 0 214_REF-KO.27B 1 -1 0 303_REF-KO.13B 1 -1 0 211_KO.18B-REF 1 1 0 300_KO.15B-REF 1 1 0 302_KO.17B-REF 1 1 0 In theory fitted values should be identical for the two coefficients (KO and WT), but in my case observed values are different (for certain genes). > fit1$coef[1:5,] KO WT [1,] -0.8372489 -0.19457588 [2,] -0.3208879 -0.15328678 [3,] -0.7202544 -0.69133039 [4,] -0.1262356 -0.08165633 [5,] -0.2568482 -0.18354345 > fit2$coef[1:5,] DyeEffect KO WT [1,] -0.411091881 -0.8372489 -0.27679426 [2,] -0.007895425 -0.3182560 -0.15328678 [3,] -0.004396641 -0.7202544 -0.69133039 [4,] 0.200046876 -0.1262356 -0.08165633 [5,] -0.052855276 -0.2568482 -0.19411451 What is the origin of these differences? Does anybody have any suggestions? Thanks again Jean-Laurent