There are different parameters to consider to end up with an optimised qPCR assay.
The MIQE Guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Bustin 2009) provide a useful overview. To make it more convenient, we have put together the most important factors in an extra guide for you:

How to estimate and compare the performance of a qPCR assay

Here in short:

  • Does the quality of my RNA match? (FragmentAnalyzer, Bioanalyzer, NanoDrop curve)
  • Test the primer efficiency with a dilution series
  • Does my amplicon match with the linear dynamic range of my assay? (5-6 step 10-fold dilution series)
  • How high is the variability between my replicates  and biological samples?
  • Does my assay produce primer-dimer or other by-products? (melting curve analysis)