Hi, The following message was not posted properly. I have analyzed a common reference with 4 groups experimental design by applying the same method described in the limma user's guide for common reference 2 groups. However, all of the reference samples were labeled with Cy5. Therefore I multiplied all of the resulted ratios with -1. Is this a valid approach? Thanks. Sohail Khan Scientific Programmer COLD SPRING HARBOR LABORATORY Genome Research Center 500 Sunnyside Boulevard Woodbury, NY 11797 (516)422-4076