Hello Bioconductorers, I have a question regarding biological v technical replication in a continuous culture model. These are very expensive experiments to carry out so are not normally repeated to get the usual biological replication required. So the following experimental design has been suggested. Cells are grown in a continuous culture fermenter and allowed to reach stasis for 5 days. At this point a sample (sample 1) is taken for analysis. Samples are taken on 2 more occasions at 3day intervals (sample 2 and sample 3). At the flow rate used, the cells present in the tank should have been completely replaced in this time period. The conditions are then changed and samples taken as indicated above - 5d for stasis to be achieved followed by sampling after sufficient time to allow for replacement of all cells (samples 4-6). We want to assess the effect of changed conditions (one only per experiment) on gene expression using BioC/affy/LIMMA. In the continuous culture model system, conditions are kept constant - levels of nutrients, metabolites etc. so the environment for each set of samples should (theoretically) be identical. My question is can we treat samples 1-3 as biological replicates and samples 4-6 as biological replicates to compare with LIMMA? Any opinions much appreciated. All the best, Peadar -- ############################ Dr. Peadar ? Gaora UCD Conway Institute, Belfield, Dublin 4. (01) 716-6915 ############################