Sorry for another anova-like question, but I have a set of two-color experiments with two variables (time (4 and 8 hours) and treatment(two levels)), but they are hybed to a common reference (untreated line). Does this design look correct (for use in limma)? Ref 4hr 8hr TrtA Trtb Treated -1 1 0 1 0 1 -1 1 0 1 0 1 -1 1 0 0 1 1 -1 1 0 0 1 1 -1 0 1 1 0 1 -1 0 1 1 0 1 -1 0 1 0 1 1 -1 0 1 0 1 1 I am interested in knowing genes differentially expressed in the treated cells, treatment A vs. treatment B, 4hr vs. 8hr. I would do contrasts something like: MakeContrasts(Treated,4hr-8hr,Trta-Trtb,levels=design) When I do the 4hr-8hr contrast, for example, does this "control" for the two different treatments? In other words, if there are different magnitude of effects due to the treatments, but the genes changing are the same at 4 and 8 hrs, will the 4hr-8hr still be significant? Thanks, Sean

At 02:19 AM 26/06/2004, Sean Davis wrote: >Sorry for another anova-like question, but I have a set of two-color >experiments with two variables (time (4 and 8 hours) and treatment(two >levels)), but they are hybed to a common reference (untreated line). Does >this design look correct (for use in limma)? No it's not. Hint: you can treat your common reference design exactly as if it was an affy experiment. See Sections 7.2 and 7.3 of the Limma User's Guide. This is a 2x2 factorial experiment. Go back through the archives of this list for answers on affy factorial experiments. One thing to remember: for an affy or common reference experiment, the number of columns in the design matrix is equal to the number of distinct groups in your experiment. You have 4 groups. Gordon >Ref 4hr 8hr TrtA Trtb Treated >-1 1 0 1 0 1 >-1 1 0 1 0 1 >-1 1 0 0 1 1 >-1 1 0 0 1 1 >-1 0 1 1 0 1 >-1 0 1 1 0 1 >-1 0 1 0 1 1 >-1 0 1 0 1 1 > >I am interested in knowing genes differentially expressed in the treated >cells, treatment A vs. treatment B, 4hr vs. 8hr. I would do contrasts >something like: > >MakeContrasts(Treated,4hr-8hr,Trta-Trtb,levels=design) > >When I do the 4hr-8hr contrast, for example, does this "control" for the two >different treatments? In other words, if there are different magnitude of >effects due to the treatments, but the genes changing are the same at 4 and >8 hrs, will the 4hr-8hr still be significant? > >Thanks, >Sean