primer desing for qPCR on de novo assembly output

Question

[pablo61991]

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 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.

[pablo61991]

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

Answer 1

[Kevin Blighe]

Hi Pablo, todo bien?

I invested a lot of time into developing a standard operating procedure (SOP) for primer design back in 2012: https://www2.le.ac.uk/centres/cancer/internal-information/procedures/sops/sop646v01.pdf

Generally, the starting point is a region of interest of ~500-1000 bases. You then have to find a target within this region of interest that is unique compared to all other sequence in the genome of the species that you are studying. This target is the target for the primers.

In my protocol, you could probably start at section 5.5, i.e, starting by finding target regions using Primer3 (http://primer3.ut.ee/) and then performing in silico PCR using the UCSC (http://genome.ucsc.edu/cgi-bin/hgPcr).

Let me know if you need further assistance!

Kevin

[pablo61991]

Thank you Kevin, I have more success using your tips ;)

[Kevin Blighe]

That's really great - glad to have helped!