Hello community,
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 donĀ“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.
Sorry but is something wrong? I can see a lot of views but no answers or comments, there is something wrong in my statement?