Dear all: I have found quite the same question being asked at least twice but I still not have clear answer about how the ebayes method in LIMMA calculate the average expression value for a given experimental group. I have used GCRMA to normalize my affimetrix values and then obtained the log2 expression values as (values below do not necessarily correspond to the same probe): 4395_CTL_LLA.CEL :7.89 4404_CTL_LLA.CEL: 8.21 4413_CTL_LLA.CEL: 8.07 I have calculated by excel: Simple mean = 8.055 Geometric mean = 8.054 Whereas the top table for average expression of these 3 values gave me: 8.055 This values are quite the same regardless of calculation method, However when more variability is among values the calculated average expression differs differs quite largely: 4368_CTL_HLA.CEL: 8.26 4394_CTL_HLA.CEL: 7.17 4400_CTL_HLA.CEL: 8.70 Simple mean = 8.042 Geometric mean = 8.015 Whereas the top table for average expression of these 3 values gave me: 8.263. In this case this average expression value seems to be the median but on the next set of samples not. 4368_CTL_HLA.CEL: 10.758 4394_CTL_HLA.CEL: 10.907 4400_CTL_HLA.CEL: 7.634 Simple mean = 8.92 Geometric mean = 9.766 Whereas the top table for average expression of these 3 values gave me: 9.862, which in this case is not at all close to the median. I will appreciate any help on this matter. It will also be appreciated, any additional though on whether the adjusted average expression (whichever the method is) is well enough to correct for expression variability within a given treatment, so I do not need to be worry for any potential outlier expression value or should I be concerned about? Regards, Miriam ******************************** Miriam Garcia, MS, PhD Department of Animal Sciences University of Florida [[alternative HTML version deleted]]