There is a lot of interest in tissue-specific and differentially expressed genes and a lot of people are using RNA-seq to determine if genes are up-regulated in one tissue vs. another. However, I am interested in a particular gene that, based on qPCR, is highly expressed in a particular aphid tissue (bacteriocyte), but it is not differentially expressed (it is also highly expressed overall in the body and in other tissues, like head and gut). So I'm not interested in using RNA-seq to measure differential expression of this gene, because I don't necessarily expect to find it. I am interested in using RNA-seq to confirm high expression in bacteriocyte (relative to other genes in the same gene family). Is there a way to do this? I know that RNA-seq does not measure absolute expression, but would it be valid to rank some measure of expression (like TPM) of, say, my gene of interest and other members of the same gene family within bacteriocyte? I would like to know if this gene and its orthologs are, or are not, highly expressed in bacteriocyte tissues relative to other genes in the same gene family. In theory, I would like to be able to quantitatively compare across orthologs/different insects, but I would be happy with independent yes/no answers for each insect.
<p>This is really a worst-case scenario for RNAseq. I would suggest that you just do qPCR, which will be both faster and vastly cheaper.</p>

<p>The issue with trying to do this with RNAseq is that there are GC and other biases that will affect the quantification of one isoform versus another. This <strong>can</strong> still be done, but you really need to be very very comfortable with the data and the underlying biases. It&#39;s much easier to do absolute quantification with qPCR, which will directly give you an answer.</p>
