I have searched in forums and google looking for help in primer design for confirmation assay by qPCR of de novo assembly transcripts. When I blast my transcripts, I have obtained different levels of identity against the blast hit and when I have to design my primers I don´t know if I'll have problems with mismatch between the assembled transcript and the real sequence.
For example, if I have a transcript for a gene A and I align my trasncript with the nucleotidic sequence of this gene A in a close related species I should design my primers in the regions with 100% identity? if these regions are not an interesting domain, how can I design confident primers for qPCR?
Maybe this is not an issue and the question is not important, in that case excuse me.
Thank you for your time.