Do you ever wonder, what is real-time PCR really gauging, is it how may target counts to start with or rather the target concentration? Let's do this experiment that fix the target concentration, while change the target counts by varying the reaction volume, and see the answer.

Fig. PCR assay mix were aliquoted into different wells with varying volume, from 5uL to 35uL. Results reveal that Ct are identical across from 5uL to 35uL reaction volume, so that Ct reflects target concentration not the target counts.