Dear list, I have a doubt about the real use of spots weights during normalization in limma. According with the documentation spots weights equal to 0 are ignored during normalization (and other posterior analyses) , and although these spots are not removed they do not have any influence on the rest. I had observed some strange behavior on my normalized data and made a tried to make a check on this. What I did was to replace weights==0 spots by NA and redo analysis. I have found the results do no spots are adequately ignored by limma or I made a conceptual mistake in this check. This is exactly the code I used for checking: > RG.b <- backgroundCorrect(RG) > MA.1 <- normalizeWithinArrays(RG.b) > RG.b$G[RG.b$weights==0] <- NA > RG.b$R[RG.b$weights==0] <- NA > MA.2 <- normalizeWithinArrays(RG.b) > MA.1$M[2,] BFN33 S50.05 S65.02 S65.03 S71.02 S75.01 S77.03 control control control control -0.96794887 0.06715693 -0.08766477 -0.50161127 -1.25216169 -0.80724650 -0.61351625 -0.80751427 -0.49960303 -0.66912129 0.27447918 > MA.2$M[2,] BFN33 S50.05 S65.02 S65.03 S71.02 S75.01 S77.03 control control control control -0.94108221 0.04803980 -0.08030398 -0.49226094 NA -0.79588160 NA -0.77182854 NA -0.61426784 0.18076174 Thank you Ana O @@@@@ Ana Conesa, PhD. @@@ O @@ O @ Centro de Gen?mica @ O @@@@ O @ Instituto Valenciano de Investigaciones Agrarias (IVIA) @@@ O @@@@ @@@@ O @ 46113 Moncada (Valencia) SPAIN || Tel. +34 963424000 ext.70161; Fax. +34 963424001 || email: aconesa at ivia.es