Quantifying Cell-Free DNA from Plasma

Plasma samples were centrifuged at 4°C at 16,000×g for 5 min to remove cell debris. The supernatant was diluted in sterile nuclease-free water at a 1:40 ratio for direct quantitative polymerase chain reaction (qPCR) measurement. The diluted plasma samples were stored at −20°C until they were analyzed. The cell free DNA (cfDNA) concentrations of the plasma were quantified using direct qPCR and a primer set (Table 1), producing an 88-bp fragment of the chromosomal myostatin (MSTN) amplicon [13 (link)]. Acting as a template, the diluted plasma was added to master mixer 20X Evagreen (SolGent, Seoul, Korea) containing 10 pmol primer set, 25 mM MgCl₂, 10 mM dNTPs, and 0.5 U BIOFACT Taq DNA polymerase (BIOFACT, Seoul, Korea). Amplification was completed over 40 cycles.