| | Re: Different cDNA volumes for target and reference genes in qPCR?
The problem is the final volume of the reaction. Commonly one prepares the master mix for ALL samples, including problem and controls. Then you add the proper volume to each tube (is the same volume for each one). In the end, after adding DNA (either genomic or complementary) template, the final volume should be the same in all tubes. Therefore, if you change the volume of template DNA you add it will ultimately change the final concentration of the master mix reagents (Mg+, dNTPs, etc.).
Since qPCR is specially sensitive, minor changes between reactions might affect the reproducibility of your results. In other words, you might have trouble infering if your results are caused by the conditions of the samples or by discrepancies between reaction tubes.
In summary, yes, it always has to have the same DNA volumes. Regarding solutions, you can mix your reference DNA in one volume of diluent (nuclease free water, TE buffer or whichever you use) so you can add 2 uL like for your target samples.