I have the following RNA-seq set-up:

- 5 samples for comparison
- matrix with TMM-normalized FPKM values

Now, TMM-normalization allows comparison across samples, so I can compare gene X between samples 1-5. Can I also compare gene X with gene Y within, say, sample 2 or does the TMM-normalization make this inappropriate (e. g. using different scaling factors for each gene or anything like that)?

I have tried to read the TMM-normalization paper (cited below) and it seems that when there are more than two samples, it picks a reference sample and calculates a single scaling factor for each pairwise comparison between the reference and each non-reference sample. That would seem to suggest that the within-sample comparison like the one outlined above is possible. Is it?

Robinson, M. and A. Oshlack (2010). "A scaling normalization method for differential expression analysis of RNA-seq data." Genome Biology 11(3): R25.
<p>TMM won&#39;t change the order of values, it&#39;s meant to adjust between samples only. So if you&#39;re asking, &quot;Does gene A have a higher FPKM than gene B in sample 3&quot;, then you&#39;ll get the same answer with/without normalization (the exact size of the difference might differ, though).</p>
