Hi, I have a question about the function normalize.loess implemented in affy package. Reading the code I understand (perhaps wrong) that the correction of the intensities of probes is done as: lg(Yi)*=lg(Yi)-1/J(Me_i1+Me_i2+...+Me_ij+...+Me_iJ) eq1) where lg stands for log2, Y is the vector of probes intensities in the slide i, J is the total number of slides, Me_ij is the estimate of M=lg(Yi/Yj) in a M vs A plot, with A=1/2lg(YiYj). This formula should come from the Dudoit et al(2002) idea of M vs A plots. For the case J=2, i.e. there are 2 slides only Eq. 1 seems corect for me since: lg(Yi)=A+M/2 eq2) lg(Yj)=A-M/2 eq3) obtained from the definition of M and A. with the normalization rule: M'=M-Me_ij i.e. Normalized log ratio M' equals raw log ratio M minus the estimated log-ratio Me_ij from the M vs A plot of the two slides i and j. If we replace the raw M in Eqs 2,3 with the normalized M', we obtain lg(Y'i)=A+M/2 - Me_ij/2 eq4) lg(Y'j)=A-M/2 + Me_ij/2 eq5) which gives eqs. 6,7: lg(Y'i)=lg(Yi) - Me_ij/2 lg(Y'j)=lg(Yj) + Me_ij/2 This matches perfectly the formula given in Eq. 1) If we treat now the case J=3: so there are three slides i, j and k Eq. 6 may be written for slide i J-1 times as there are J-1 pairs: lg(Y'i)=lg(Yi) - Me_ij/2 lg(Y'i)=lg(Yi) - Me_ik/2 summing the J-1 equations and dividing by J-1: we obtain Eq. 10: lg(Y'i)=lg(Yi) -(Me_ij+Me_ik)/2/(J-1) As you may see this formula matches perfectly Eq 1 if there are only two slides (J=2) but diverges for J>2. Has anyone an idea what is wrong in all this? Am I doing something wrong or normalize.loess is incorrect? Thanks, Laurentiu Tarca