Normalization for two bulk RNA-Seq samples to enable reliable fold-change estimation between genes I have two bulk RNA-Seq samples, already tpm-normalized.

I would like to know what is a reasonable normalization procedure to enable downstream log fold-change estimation.

The distribution of the two samples using the common set of genes looks similar:



However, the two samples have only been tpm-normalized, is it enough to guarantee reliable fold-change estimation? Should I use another normalization procedure, e.g. Quantile Normalization, before comparison?

My objective is to define a signature using the genes that are up-regulated in Sample1 with respect to Sample0, and vice versa. I'm using log fold-changes, but I'm concerned that their value may be affected by each sample distribution.

Do you also have suggestions for the definition of up-regulated genes with these data?
You have only two samples?

You aren't going to be able to draw strong conclusions from that no matter what you do.  Clever statistics don't work without replicates.