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:
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)