Dear list, It might be a really easy problem but I can not figure out how to do it. I'm trying to analyze a set of hybs of a time course experiment, where I used a loop design. I have six samples and no reference, I compare (1-2,2-3,3-4,4-5,5-6,6-1); each with dye swaps so I have 12 slides. By selecting sample 1 as my reference in modelMatrix, I get the following design matrix: 2 3 4 5 6 1 0 -1 1 0 0 2 0 0 -1 1 0 3 0 0 0 -1 1 4 1 0 0 0 -1 5 0 1 0 0 0 6 0 0 0 1 -1 7 0 1 -1 0 0 8 -1 0 0 0 1 9 1 0 0 0 0 10 0 0 1 -1 0 11 0 -1 0 0 0 12 -1 0 0 0 0 My question is: How to use contrastMatrix to obtain the following comparisons: 1-2, 2-3, 3-4, 4-5, 5-6 and 6-1? I know there is some information about Limma and time course experiments but that example uses one-color arrays so I'm confused about how to apply that to my experimental design. Thanks in advance for any help, Sergio