Gene set analysis needs to have a set of genes. It isn't possible to perform gene set analysis on the expression data of only one gene. The typical process is to have two groups of samples (could be your EGFR HI/LOW groups), perform differential expression on ALL genes, and then do Gene Set Analysis on the resulting differentially-expressed genes.

if I get your question right...you divided the patients in EGFR high group and EGFR low group. this means that you have expression levels for ALL genes in these two groups. you are now trying to use GSEA to evaluate the enrichment of some signatures (i.e. HALLMARK or KEGG or whatever) and see if there is a difference between the two groups that you created. correct?

if that's the case, yes you can/could, but I think there are a couple of caveats. GSEA itself was designed for microarray data while you have RNASeq data (I'd guess). you can normalize/analyze your data for GSEA as described here. there is also a Bioconductor package called SeqGSEA that might be closer to what you look for.

personally I think that if you normalize and transform your data correctly (and don't use FPKM) you should be fine using those data as input for GSEA.

hope this helps

Thanks Sean,

sorry I forgot to mention that I have third file also (C2: curated gene sets downloaded from msigdb)

But how can I perform differential expression for all gene basis of EGFR HI/LOW.

Is it possible or not?

Thanks

Thanks TriS,

Yes absolutely you got my question, my data is mRNA Expression z-Scores (RNA Seq V2 RSEM), and I divided tumor samples based on high EGFR and Low EGFR, BUT I not included all genes, I have only EGFR gene. Is it possibe? or I should include all genes.

Thanks,