Dear list, I am analyzing a two color microarray dataset using limma. The dataset is from ArrayExpress database (accession number E-MEXP-1949). The samples include 4 mutants (rcd11, rcd13, rcd14 and sro11) and wild- type Col0. For the 4 mutants there are 4 biological replicates each. For Col0 there are also 4 biological replicates but for each biological replicate there are 4 technical replicates. The targets I made is as the following: > targets FileName Cy3 Cy5 1 122.rcd1.gpr.proc rcd11.r1 Col0.r1 2 124.354.gpr.proc sro11.r1 Col0.r1 3 125.466.gpr.proc rcd14.r1 Col0.r1 4 126.500.gpr.proc rcd13.r1 Col0.r1 5 133.rcd1.gpr.proc rcd11.r2 Col0.r2 6 134.354.gpr.proc sro11.r2 Col0.r2 7 135.466.gpr.proc rcd14.r2 Col0.r2 8 136.500.gpr.proc rcd13.r2 Col0.r2 9 140.90506.0001.gpr.proc Col0.r3 rcd11.r3 10 141.090506.0001.gpr.proc Col0.r3 rcd14.r3 11 142.090506.0001.gpr.proc Col0.r3 rcd13.r3 12 143.090506.0001.gpr.proc Col0.r3 sro11.r3 13 152.110506.0001.gpr.proc Col0.r4 rcd14.r4 14 153.110506.0001.gpr.proc Col0.r4 rcd13.r4 15 154.110506.0001.gpr.proc Col0.r4 rcd11.r4 16 155.110506.0001.gpr.proc Col0.r4 sro11.r4 I am interested in finding DE genes between different mutants (rcd11...) and wild-type Col0. Reading in the data and normalization have been done but I was not able to make a design matrix. I have read the whole limma users guide, especially the part dealing with technical replicates but could not find something that is similar to this. Thanks in advance for your help! Yong Li