Dear Bioconductors, I known that this mailing list is about bioconductor software, but, I read a few vignetes and, after this, I have a doubt about my future experimental design. I am sorry for this. We biological experiments is: A bacterium parasite of plants in its wild type state will be compared with a mutant in three different times (0, 1, and 3 days after the inoculation). In the time zero, bacterial cells, wild type and mutant, they will be inoculated in its host. In the all times above, the RNA of each one of them will be collected. Soon, for each time we will have RNA of both, of the wild type and of the mutant. MT1 x WT1 MT2 x WT2 MT3 x WT3 Our doubt is: which experimental design to use? For example: This one? RNA mutant in T1 compared to RNA mutant in T2 compared to RNA mutant in T3 compared to RNA mutant in T1 (loop) MT1 MT3 MT2 Or this one? One pool of wild type RNA would be used as reference (control)? In this case, RNA mutant in MT1 will be compared to WT RNA pool, RNA mutant in MT2 will be compared to WT RNA pool, and RNA mutant in MT3 will be compared to WT RNA pool. MT1 MT2 WT (pool) MT3 Or we compare, in each one of the times, the mutant RNA versus the wild type RNA and later we proceed the identification the difference of expression between the times using, for example, limma? MT1 MT2 MT3 x x x WT1 WT2 WT3 We are interested in verifying the differential expression to the long one of the time. I ask a statistician here, but he dont have expertise for this situation. Any suggestion is very apreciate. Thansk a lot Marcelo