Hi all, I am analyzing an experiment with two-color arrays in which expression data was collected from two genotypes in two different environments according to a loop design. I wanted to use LIMMA to test for effects due to genotype, environment, and their interaction. The LIMMA user's guide has a good example on a similar factorial design which includes, in addition to the four treatments of interest, an external fifth RNA source used as a reference. I don't have such a reference in my design and am having a hard time coding for the design matrix such that I can make the contrasts between genotypes (1&2), between environments (1&2), and also have an term for the interaction of genotypes and environments. Below is a scheme of the design, with lines being dye-swap hybridizations: genotype_1_Environment_1 ---------- genotype_1_Environment_2 | | | | | | | | genotype_2_Environment_1 ---------- genotype_2_Environment_2 Any ideas on how to code for the design matrix in such a case? Thanks, Bernardo Lemos.