Primer design to do qPCR on repetitive elements

Question

[Gema Sanz]

Hi,

I need to design qPCR primers to amplify some human repetitive elements. I'm using Primer BLAST and I wonder if there are any specific parameters to take into account to design them, i.e., Specificity check, Database... I removed the repeat filter but I'm not sure that the primer pair that I'm getting are ok since they are not working in UCSC In-Silico PCR...

Any help or suggestion will be very much welcome!

Best Gema

Answer 1

[trausch]

Silica can check primers genome-wide. It uses the same thermodynamic calculations as Primer3 and outputs all primer binding sites and possible amplicons. For a repetitive element it's probably a bit back and forth of designing new primers using slightly different parameters and checking them until you find a unique amplicon.

[Gema Sanz]

Thanks! I'm not looking for an unique amplicon, I want to amplify all the possible locations that can contribute to expression, I'm using Repbase consensus sequence, but somehow when checking all the possible matches with in silico PCR, for some repeats I don't get any results.