Isolation of cell-free DNA from cell culture supernatant: Method comparison

In vitro characterization of cell-free DNA (cfDNA) represents an important step in pursuit of developing a better understanding of the physical and biological characteristics of cfDNA in vivo. However, precise measurement of the cfDNA present in cell culture medium is highly dependent on the efficacy of the DNA isolation method, and is often a juncture of experimental confusion. Therefore, in this study we compared six of the most commonly used and commercially available cfDNA isolation kits for the recovery of cfDNA from the cell culture supernatant of human bone osteosarcoma (143B) cells, including the (i) QIAamp circulating nucleic acid kit, (ii) cfPure cfDNA extraction kit, (iii) Magmax cell-free DNA isolation kit (manual protocol), (iv) NucleoSpin Gel and PCR clean-up kit, (v) NucleoSpin plasma XS kit, and (vi) MagNa Pure 24 system. Based on cfDNA quantification and size evaluation, using the Qubit dsDNA HS assay and Bioanalyzer HS DNA assay, respectively, the different methods have shown marked variability concerning cfDNA yield, reproducibility, as well as size discrimination. Regardless of overall yield, all extraction methods based on the purification of cfDNA using magnetic beads recovered a higher fraction of small cfDNA fragments (50-250 bp) compared to extraction methods based on the binding of cfDNA to silica-based membranes. This highlights the importance of selecting a cfDNA isolation method that is appropriate for the aims of a study. For example, mutational analysis of cfDNA may be enhanced by the selection of a cfDNA extraction method that favors a high yield, and is biased toward the isolation of small cfDNA fragments (e.g., 50 – 250 bp). In contrast, quantitative analysis of cfDNA in a comparative setting (e.g., measuring the fluctuation of cfDNA levels over time) may require the selection of a cfDNA isolation method that forgoes a high recovery for high reproducibility and minimal size bias.