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 [[alternative HTML version deleted]]