I recently conducted an experiment and there seems to be quite some variation within replicate groups. The experiment was done to high standard and the extraction of RNA was done all the same way on the same day to eliminate the possibility of batch effects. I used 3 replicates ( more reps prob would have been best in hindsight!).Samples were from wild type organisms from the field, which were acclimated for several weeks in the same conditions. I know gene expression is stochastic, but it seems this data set is rather tricky.
Has anyone got any experience of working with variability and dispersion within replicate groups and can point me in the right way of a few methods? I've ran EdgeR and the model does not really fit well. I am running a single factor experiment.. I get D.E, but it is much less than what I thought,especially for the magnitude of difference between treatments.
Any ideas or ways I can go about analysis? Outliers I guess are somewhat tricky to call when you have a low amount of reps; however, there is 1 outlier in one replciate group by the looks of it. Is it normal to remove outliers in RNAseq?