A Comparison of Cell-Free DNA Isolation Kits: Isolation and Quantification of Cell-Free DNA in Plasma

The analysis of cell-free DNA (cfDNA) as a sensitive biomarker for cancer diagnosis and monitoring has resulted in a need for efficient and standardized cfDNA isolation. In this study, we compared the isolation efficiency of the QIAamp circulating nucleic acid kit (QIA) with four other cfDNA isolation kits: the PME free-circulating DNA Extraction Kit (PME), the Maxwell RSC ccfDNA Plasma Kit (RSC), the EpiQuick Circulating Cell-Free DNA Isolation Kit (EQ), and two consecutive versions of the NEXTprep-Mag cfDNA Isolation Kit (NpM_V1/2). cfDNA was isolated from 10 plasma samples, of which five contained KRAS mutated cell-free tumor DNA (ctDNA). Digital droplet PCR was used to quantify the total cfDNA concentration as well as the KRAS mutated ctDNA fraction. cfDNA integrity was assessed with real-time quantitative PCR. The QIA and the RSC kits displayed similar isolation efficiencies of both KRAS mutated ctDNA and nonmutated cfDNA, whereas the yield generated by the PME and NpM_V2 kits was significantly lower. Real-time quantitative PCR indicated the presence of digital droplet PCR inhibiting agents in the eluates of the NpM_V1 and EQ kits. To conclude, this study presents two highly efficient isolation kits for cfDNA isolation, of which the RSC kit has the advantage of a fully automated protocol over the labor-intensive QIA kit.