A phenol-chloroform free method for cfDNA isolation from cell conditioned media: development, optimization and comparative analysis

Abstract

The non-invasive invasive nature of cell-free DNA (cfDNA) as diagnostic, prognostic, and theragnostic biomarkers has gained immense popularity in recent years. The clinical utility of cfDNA biomarkers may depend on understanding their origin and biological significance. Apoptosis, necrosis, and/or active release are possible mechanisms of cellular DNA release into the cell-free milieu. In-vitro cell culture models can provide useful insights into cfDNA biology. The yields and quality of cfDNA in the cell conditioned media (CCM) are largely dependent on the extraction method used. Here, we developed a phenol-chloroform-free cfDNA extraction method from CCM and compared it with three others published cfDNA extraction methods and four commercially available kits. Real-Time PCR (qPCR) targeting two different loci and a fluorescence-based Qubit assay were performed to quantify the extracted cfDNA. The absolute concentration of the extracted cfDNA varies with the target used for the qPCR assay; however, the relative trend remains similar for both qPCR assays. The cfDNA yield from CCM provided by the developed method was found to be either higher or comparable to the other methods used. In conclusion, we developed a safe, rapid and cost-effective cfDNA extraction protocol with minimal hands-on time; with no compromise in cfDNA yields.