I have used the TopHat-Cufflinks-Cuffdiff pipeline to carry RNAseq analysis (looking at differential gene expression).
The experiment has been carried in two donors, and twice for each donor giving a total of 4 replicates for each condition. There are 5 conditions and therefore 20 samples in total that have bee sequenced.
After using cuffdiff we found that we do not see genes significantly differentially expressed (according the corrected q value that the software calculates). The only significant results (q < 0.05) that I see are for one of the conditions- and this is the case for less than 20 genes.
Can anyone suggest how i can interpret my data/ re-think how to analyse. Perhaps using packages that take into account biological variation? (My guess is that variation in my sample is affecting the p/ q values. I have run cufform for all samples- the FPKM values for replicates are quite variable). Or can I use the p values or the fold changes to inform of genes that are differentially expressed between samples?