Hi, We are currently planning a timecourse experiment, which will be analysed using Limma, and would be grateful for some advice on the design matrix we have come up with... Sorry, it's quite complicated and may take some figuring out if I haven't explained it as well as I might........ The experiment consists of cells teated with 2 different drugs, with samples taken after 3 different times after addition of drug. Instead of the simple possibility of comparing everything back to the untreated t0 sample, we are currently favouring making a loop out of the untreated samples (t0, t1, t2, t3), then comparing the two treatments at each timepoint to the untreated sample at that timepoint. All comparisons will be in both dye orientations. The possible design matrix we have is as follows- Slide Untreated Drug1 Drug2 t1-t0 t2-t1 t3-t2 t1-t0 t2-t1 t3-t2 t1-t0 t2-t1 t3-t2 1 1 0 0 0 0 0 0 0 0 2 -1 0 0 0 0 0 0 0 0 3 0 1 0 0 0 0 0 0 0 4 0 -1 0 0 0 0 0 0 0 5 0 0 1 0 0 0 0 0 0 6 0 0 -1 0 0 0 0 0 0 7 -1 0 0 1 0 0 0 0 0 8 1 0 0 -1 0 0 0 0 0 9 -1 0 0 0 0 0 1 0 0 10 1 0 0 0 0 0 -1 0 0 11 -1 -1 0 1 1 0 0 0 0 12 1 1 0 -1 -1 0 0 0 0 13 -1 -1 0 0 0 0 1 1 0 14 1 1 0 0 0 0 -1 -1 0 15 -1 -1 -1 1 1 1 0 0 0 16 1 1 1 -1 -1 -1 0 0 0 17 -1 -1 -1 0 0 0 1 1 1 18 1 1 1 0 0 0 -1 -1 -1 We hope that this will give us after linear model fitting data corresponding to the separate timecourses for the untreated and 2 sets of treated cells - have we done this sensibly? Many thanks in advance for any advice at all, Gareth --