I have Illumina RNAseq data and want to look for differences in gene expression between male and female rats and transgenic vs. wildtype; for each condition I have triplicates. I mapped with TopHat for Illumina, using the reference genome rn5 and default settings. I did Cuffdiff afterwards and used the GTF-file Rattus norvegicus.Rnor_5.0.72.gtf as transcript. As result I got no significant changes in expression and it always says "NO TEST" (or sometimes "LOW DATA"). I found that lowering or raising the -c option might control the cuffdiff behavior, but I do not know what it is and how to control it. Can you explain me how to do it? And would you advise me to use another transcript for Cuffdiff? And when yes, where can I get it?
Lots of questions. Thanks in advance for your answer.