I would appreciate advice on how to construct a contrast matrix for a 5X2 ANOVA design. Briefly, I have a genomic experiment to analyze that compares 5 brain regions in 2 strains of rats. We are interested in discovering overall differences between strains (collapsing all brain regions together) but also discovering differences that may only be expressed in one brain region. I have attempted to construct the appropriate matrix with the code listed below, but it does not work. I seem to get differences between strains, but all the brain region contrasts give exactly the same results, so I know something isn't correct. contrast <-makeContrasts( ( (NPAccumbens + NPAmygdala + NPHippocampus + NPPrefrontal_Cortex + NPStriatum) - #all regions of strain "NP" (PAccumbens + PAmygdala + PHippocampus + PPrefrontal_Cortex + PStriatum) #all regions of strain "P" ), (NPAccumbens - PAccumbens), #accumbens region of both strains (NPAmygdala - PAmygdala), #amygdala region of both strains (NPHippocampus - PHippocampus), #hippocampus region of both strains (NPPrefrontal_Cortex - PPrefrontal_Cortex), #Prefrontal_Cortex region of both strains (NPStriatum - PStriatum), #striatum region of both strains levels=design) Thanks! Mark Mark W. Kimpel MD (317) 490-5129 Home, Work, & Mobile (317) 278-4104 FAX