Dear All, As it described in Limma User's Guide chapter 8.7 there are more than one approach to create the design and contrst matrices. I would like to focus in this question on the first example where in the design matrix one creates all possible combinations between the factors. Thus for target file of Genotype and treatment FileName Strain Treatment File1 WT U File2 WT S File3 Mu U File4 Mu S File5 Mu S we get a design matrix as follow WT.U WT.S Mu.U Mu.S 1 1 0 0 0 2 0 1 0 0 3 0 0 1 0 4 0 0 0 1 5 0 0 0 1 attr(,"assign") [1] 1 1 1 1 attr(,"contrasts") attr(,"contrasts")$TS [1] "contr.treatment" and contrast for the interaction is Int WT.U 1 WT.S -1 Mu.U -1 Mu.S 1 My question is how to move to three factors. I know that this design was pointed by Sean in one of the previous messages. However, he was intrested mainly in the main effects and the solution that was suggested by Gordon was based on the "treatment-contrast parametrization" approach by running model.matrix(~factorA*factorB*factorC). I would like to use the "all combinations" approach and I have the following experimental design Dev Treat Cultivar 1 Stage1 Ctl HS 2 Stage1 Str HS 3 Stage1 Ctl HS 4 Stage1 Str HS 5 Stage1 Ctl HT 6 Stage1 Str HT 7 Stage1 Ctl HT 8 Stage1 Str HT 9 Stage2 Str HS 10 Stage2 Ctl HS 11 Stage2 Str HS 12 Stage2 Ctl HS 13 Stage2 Ctl HT 14 Stage2 Str HT 15 Stage2 Ctl HT 16 Stage2 Str HT after creating a design matrix by DTC <- factor(paste(ThreeWay$Dev, ThreeWay$Treat, ThreeWay$Cultivar, sep=".")) design<-model.matrix(~0+DTC) I am interesting in in three interactions. The interaction with in each stage between Treatment and Cultivar and Interaction between all, i.e., Dev*Treat*Cultivar. Would the contrasts matrix for these question should be looks like that Stg1.Tr.Cult Stg2.Tr.Cult Stg.Tr.Cult Stage1CtlHS 1 0 -1 Stage1CtlHT -1 0 1 Stage1StrHS -1 0 1 Stage1StrHT 1 0 -1 Stage2CtlHS 0 1 1 Stage2CtlHT 0 -1 -1 Stage2StrHS 0 -1 -1 Stage2StrHT 0 1 1 ? Thanks, Ron