Development of an Effective Method to Enrich Cell-Free Nucleic Acids in Liquid Samples

We aimed to establish a method to extract cell-free DNA (cfDNA) from liquid samples by combining protamine solution with nucleic acid extraction reagents.

Samples comprised 1 mL EDTA/2K plasma from healthy individuals (n = 22) and 5, 10, 20, and 40 mL K562 cell culture supernatants. After adding protamine solution and NaCl, the samples were incubated and subsequently centrifuged. Genomic DNA (gDNA) was extracted using a High Pure PCR Template Preparation Kit. For comparison, plasma was extracted using a cobas® cfDNA Sample Preparation Kit. The gDNA was subjected to real-time quantitative PCR.

Our method yielded results comparable to those obtained using a cfDNA-specific kit. Culture supernatants showed volume-dependent yield variations, and gDNA was successfully extracted from samples of all volumes following enrichment.

Our method enhanced cfDNA enrichment, highlighting its potential for seamless integration with commonly available nucleic acid extraction reagents to process large sample volumes.