Proper reference set selection in enrichment analysis

Question

[lentram2]

Dear all,

I performed a GO enrichment analysis (Fisher’s Exact Test; FDR<0.05; Pvalue<0.001) in B2G using as test set the up-regulated differentially expressed genes (DEGs) in each comparison of each treatment meanwhile the whole set of DEGs of the experiment were considered as reference set.

I believe that if I would like to know the significant functional characteristics of the DEGs of each comparison, as I did should be OK. But, should I have performed the enrichment analysis to the overall transcriptome (as reference set) in order to increase the power of the test ??

Any advise would be highly appreciate!!

Thanks so much

Answer 1

[WouterDeCoster]

Your reference set should consist of every gene for which a measure was obtained in the experiment, for expression that means every gene which was detected (perhaps above a certain minimal read cutoff). Using only the differentially expressed genes doesn't sound like the way to go.