Dear Carthika, This is a somewhat special design, and so isn't specifically covered by the limma User's Guide. It's like a direct design for LTP, but like a common reference design for comparing the grous. I suggest agegroup <- factor(targets$Name) design <- model.matrix(~0+agegroup) colnames(design) <- levels(agegroup) Then the three coefficients compare LTP to Control within each of the three age groups. To compare the age groups, just extract contrasts as usual, cont.matrix <- makeContrasts(YvsA=young-aged,levels=design) and so on Best wishes Gordon > Date: Thu, 20 Nov 2008 12:13:13 +1300 > From: Carthika Luxmanan <carthika.luxmanan at="" anatomy.otago.ac.nz=""> > Subject: [BioC] Creating a model matrix for three age groups using > two-colour arrays > To: bioconductor at stat.math.ethz.ch > Content-Type: text/plain > > Hi > > I am new to Bioconductor R/Limma. I've worked through some examples, > and familiarized myself with some basic commands. However, I am not > sure how to create a model matrix for my experiment. We looked at > three age groups of rats (YA, MA and OA) to look at gene expression > changes following LTP (LTP is a memory model). LTP was stimulated on > one side of the brain, while the opposite side served as control. So > RNA from Control side was coupled to Cy3, while RNA from LTP side was > coupled to Cy5. A single microarray was used for each animal, with n=5 > for YA, 3 for MA and 5 for OA. My Targets file looks as follows: > > SlideNumber Name FileName Cy3 Cy5 > 1 young YA19_with norm_15Sept06.gpr Ct LTP > 2 young YA23_with genepix norm.gpr Ct LTP > 3 young YA 24_with norm.gpr Ct LTP > 4 young YA27_with norm_15Sept06.gpr Ct LTP > 5 young YA28_with norm_15Sept06.gpr Ct LTP > 6 middleaged MA3_with norm_15Sept06.gpr Ct LTP > 7 middleaged MA12_with norm_15Sept06.gpr Ct LTP > 8 middleaged MA15_with norm_15Sept06.gpr Ct LTP > 9 aged OA14_with norm.gpr Ct LTP > 10 aged OA 19_with norm.gpr Ct LTP > 11 aged OA 32_with genepix norm.gpr Ct LTP > 12 aged OA37_with norm_150906.gpr Ct LTP > 13 aged OA38_bal_15Sept06.gpr Ct LTP > > How can I differentiate the groups in a model matrix? I would like to > compare the gene expression changes between the three groups. I would > have liked to look at the gene expression changes as a result of LTP > in the individual groups too, but I have been warned recently that > this might bring up more false positives since I do not have dye swaps > within each group. > > Thanks in advance. > > Carthika