Dear all I would greatly appreciate any help with the following. We have recently conducted a microarray experiment to investigate the effect of amplification on expression ratios. The design of the experiment is as follows: group1: DRG amplified RNA (4 Affymetric replicates) group2: spinal cord amplified RNA (4 Affymetrix replicates) group3: DRG unamplified RNA (3 Affymetrix replicates) group4: spinal cord unamplified RNA (3 Affymetrix replicates) I fed all data through to limma and specified the following contrasts: contrast1: group1-group2 contrast2: group3-group4 contrast3: (group1-group2) v (group3-group4) I began by examining the level of concordance between contrast 1 and 2 so I cross compared the top 100 (....going up to top 1000)genes from contrast 1 and 2 looking for the extent of agreement between them. The results are quite nice showing in average more than 80% agreement. However, if I only feed group1 and group2 to limma without the rest of the data and repeat contrast1. And separate to that, feed group 3 and group 4 to limma and repeat contrast2 the percentages are very different and vary with the number of genes selected so as an example no top genes %agreement between contrast1 and 2 100 27 300 40 500 50 2000 60 I have a feeling that the former is more correct statistically speaking but I can't say for sure what is correct and justify the difference between the two observations. I tried to read more about the statistical theory of limma but it seems well above my modest understanding of statistics. Can somebody help me resolve this confusion. In particular, I want to know which of the two approaches is more sensible and why is causing the difference between them. NS: all chips are Affymetrix Many thanks Regards