Hello, This is my first time to past my help here, after I got lost from a couple of requests. As suggested by professor Gordon Smyth, I am writing to get some help on LIMMA package while I am trying to analyze my dual-dye microarray data. Before wrote this email I have I tried following LIMMA guide but still have several places unclear with LIMMA for my data. The first part of my experiment consists of a loop design to compare the gene expression of at different development stages (10DAP, 22DAP and 35DAP, day-after-pollenization) of the same Brassica line. I have dye swap plus two technical replicates, here is the targets file: FileName Cy3 Cy5 AT Oligo 02.11.02.176.gpr.fixed 10DAP 22DAP AT Oligo 02.11.02.177.gpr.fixed 22DAP 10DAP AT Oligo 02.11.02.178.gpr.fixed 22DAP 10DAP AT Oligo 02.11.02.179.gpr.fixed 10DAP 22DAP AT Oligo 02.11.02.180.gpr.fixed 22DAP 35DAP AT Oligo 02.11.02.181.gpr.fixed 22DAP 35DAP AT Oligo 02.11.02.182.gpr.fixed 35DAP 22DAP AT Oligo 02.11.02.183.gpr.fixed 35DAP 22DAP AT Oligo 02.11.02.184.gpr.fixed 10DAP 35DAP AT Oligo 02.11.02.185.gpr.fixed 10DAP 35DAP AT Oligo 02.11.02.186.gpr.fixed 35DAP 10DAP AT Oligo 02.11.02.187.gpr.fixed 35DAP 10DAP This experiment is very similar to the design in LIMMA User's Guide section 7.4, except I have technical replicates. From the Guide, should I have to use one sample like "10DAP" as reference, or any sample for a reference? My goal is to see which genes are differentiated from 10DAP, 22DAP and 35 DAP. How do I get the results of: 1)which genes are consistently up/down-regulated across the 3 stages? 2) which genes are up-down-regulated at each development stage? The second part of my experiment is FileName DPA Cy3 Cy5 2009-07-10-atq3.7.3.145-15.gpr 15DPA WT MUTANT 2009-07-15-atq3.7.3.146-15.gpr 15DPA MUTANT WT 2009-07-15-atq3.7.3.147-15.gpr 15DPA MUTANT WT 2009-07-15-atq3.7.3.148-15.gpr 15DPA WT MUTANT 2009-07-15-atq3.7.3.149-15.gpr 15DPA WT MUTANT 2009-07-17-atq3.7.3.151-20.gpr 20DPA MUTANT WT 2009-07-17-atq3.7.3.152-20.gpr 20DPA WT MUTANT 2009-07-17-atq3.7.3.153-25.gpr 25DPA MUTANT WT 2009-07-17-atq3.7.3.154-25.gpr 25DPA WT MUTANT 2009-07-17-atq3.7.3.155-10.gpr 10DPA MUTANT WT 2009-07-17-atq3.7.3.156-10.gpr 10DPA WT MUTANT 2009-07-17-atq3.7.3.157-30.gpr 30DPA MUTANT WT 2009-07-17-atq3.7.3.158-30.gpr 30DPA WT MUTANT 2009-07-21-atq3.7.3-159-10.gpr 10DPA MUTANT WT 2009-07-21-atq3.7.3-160-20.gpr 20DPA MUTANT WT 2009-07-21-atq3.7.3-164-25.gpr 25DPA MUTANT WT 2009-07-21-atq3.7.3-256-30.gpr 30DPA MUTANT WT 2009-07-22-atq3.7.3-115-10.gpr 10DPA MUTANT WT 2009-07-22-atq3.7.3-116-20.gpr 20DPA MUTANT WT 2009-07-22-atq3.7.3-117-25.gpr 25DPA MUTANT WT 2009-07-22-atq3.7.3-118-30.gpr 30DPA MUTANT WT 2009-07-22-atq3.7.3-119-10.gpr 10DPA WT MUTANT 2009-07-22-atq3.7.3-120-20.gpr 20DPA WT MUTANT 2009-07-22-atq3.7.3-124-25.gpr 25DPA WT MUTANT 2009-07-22-atq3.7.3-125-30.gpr 30DPA WT MUTANT This is a time course experiment. Again I want to see the expression differentiation across the stage (10, 15 20, 25 and 30DAP) . Can I split the analysis into five sub-groups instead of a whole? If I split the 25 slides into 5 sub-experiments, my feeling is the variance and normalization would be different from each other. Is this correct? How do I prepare the biolrep as I do not have any biological replicates? I would appreciate very much if you could give me some suggestions on these questions. Thank you very much for your help! Sincerely yours, Yifang Tan [[alternative HTML version deleted]]