There are many factors to be considered – and to be tested – to answer this question.

Please be aware that too much template as well as products of the reverse transcription process may inhibit the qPCR reaction. We thus strongly recommend to start with a 5-6 step 10-fold dilution series (e.g. 0.001 ng, 0.01 ng, 0.1 ng, 1 ng, 10 ng) to determine the optimal amount of template for your setup.

A dilution series allows to determine the following parameters:

  • Efficiency of primers
  • Inhibition of the qPCR reaction by template/rT-products
  • The amount of template needed to run the qPCR in the linear slope

It is important to adjust the amount of template in a way that the qPCR reaction runs in the linear part of the amplification curve. Typically this is the case with in between 1 ng and 10 ng template/reaction.