I've recently designed a few primers by using NCBI's Primer-BLAST for RT-PCR and did not notice that one of the potentially unintended transcripts is the non-protein coding variant transcript (XM_) of the targeted gene. I have already ordered the primers and so I was wondering what I can do to tell apart the protein and non-protein coding transcript RT-PCR results. So far I've thought of just running a gel.
Andrei S. Babenka
Dear Andy, first of all align all transcript variants from NCBI including XM and NM or at least your target NM with XM. If your sequence the same in 100% - you can't discriminate it by PCR or electrophoresis. But if you have some differences in amplicon length - it is great. In this case you can use gel.
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