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.