Partly in response to Anthony's question and to further the low rep affy discussion I'd like to get more views on this. I have 3 paired biological reps of untreated (U) and treated (T), normalised by RMA and in this case looked at 2 fold changes. I made the U-T pairwise comparison for each replicate and counted the number of genes where fold change >2. To assess false positives I compared within the biological replica 1-2, 1-3 and 2-3 for genes >2. I got the following results. 1rep 2rep 3rep U-T 1544 1201 1008 These are the changes due to treatment U 31 13 1 False positives? T 66 27 4 " The biological replica seem to be very good, and the within replica comparisons suggest very low false positive rates. However the inclusion of an additional replica loses c.200-300 genes in the U-T, whilst the within replica comparisons suggest much lower false positives. I guess this is because no account of variance has been taken and there will be many genes with distributions around the 2fold change for U-T, but any other comments on this approach? If you were a reviewer what would be your comments on assessing false positive rates by comparing between biological replica versus comparing U-T? Thanks Matt