The following parameters determine the specifity of a qPCR assay
- Primer concentration
- Annealing temperature and the melting temperature of the primers
- Annealing time
- Occurence of primer-dimer
What can you do if your qPCR shows a lack of specifity?
Decrease the primer concentration
Increase the annealing temperature – but do not exceed the melting temperature of the primers!
Reduce the annealing time. That makes the annealing of the primers more stringent so that primers with the best match mostly make the race.
Design new primers with higher melting temperature and optimal length.
Check the specifity of your qPCR via agarose gel, or – in case you use SYBRGreen – via the melting curve.