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

摘要

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 (NpMv(1)/v(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 NpMv(2) kits was significantly Lower. Real-time quantitative PCR indicated the presence of digital droplet PCR inhibiting agents in the eluates of the NpMv(1) 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.