Hi! I am looking for advice on how to get lists of differentially expressed genes for my experiments. Generally I have four plant lines (wt and 3 mutant lines) and two different conditions. But I do not have one common reference. I always compared treated plants to untreated pool of the same line and additonally I compared untrated mutants to wt pool. I performed 4 replicates of each (biological replicates with respect to Cy5 channel). Background correction and normalization are done. These are my arrays: Cy3 Cy5 pool.wt wt.cond1 pool.wt wt.cond1 pool.wt wt.cond1 pool.wt wt.cond1 pool.wt wt.cond2 pool.wt wt.cond2 pool.wt wt.cond2 pool.wt wt.cond2 pool.mu1 mu1.cond1 pool.mu1 mu1.cond1 pool.mu1 mu1.cond1 pool.mu1 mu1.cond1 pool.mu1 mu1.cond2 pool.mu1 mu1.cond2 pool.mu1 mu1.cond2 pool.mu1 mu1.cond2 pool.mu2 mu2.cond1 pool.mu2 mu2.cond1 pool.mu2 mu2.cond1 pool.mu2 mu2.cond1 pool.mu2 mu2.cond2 pool.mu2 mu2.cond2 pool.mu2 mu2.cond2 pool.mu2 mu2.cond2 pool.mu3 mu3.cond1 pool.mu3 mu3.cond1 pool.mu3 mu3.cond1 pool.mu3 mu3.cond1 pool.mu3 mu3.cond2 pool.mu3 mu3.cond2 pool.mu3 mu3.cond2 pool.mu3 mu3.cond2 pool.wt mu1.U pool.wt mu1.U pool.wt mu1.U pool.wt mu1.U pool.wt mu2.U pool.wt mu2.U pool.wt mu2.U pool.wt mu2.U pool.wt mu3.U pool.wt mu3.U pool.wt mu3.U pool.wt mu3.U I am interested in: performing all simple comparisons Cy5/Cy3 find difference in response to cond 1 and cond2 for each separate line find differences in response to cond1 between all lines, the same for cond2 find differences between untreated mutant lines. Can I do it by designing one smart matrix and making contrasts? or shall I better divide my tasks, for example into groups with one common reference each and perform the comparisons separately? But how do I get the combined contrasts then? As I mentioned, I am a begginer so I can try to follow the examples in the manual but combining them somehow seems too much for me... Any help is welcome :-) One more question: I filtered out some bad spots and the values are set no NA. Will they be simply ignored and only existing values from other array replicates will be combined for a given gene? Hope so. I know that giving spot weights would be better but that's left for the future... Dr Agnieszka ?mie?ko Centrum Doskonalosci CENAT Instytut Chemii Bioorganicznej Polskiej Akademii Nauk Noskowskiego 12/14 61-704 Pozna? tel. (61) 8528503 wew. 249 fax: (61) 8520532 Agnieszka Zmienko, Ph.D. CENAT Institute of Bioorganic Chemistry Polish Academy of Sciences Noskowskiego 12/14 61-704 Poznan, Poland phone (0048) 61-8528503 ext. 249 fax: (0048) 61-8520532