Effects of Quantification Methods, Isolation Kits, Plasma Biobanking, and Hemolysis on Cell-Free DNA Analysis in Plasma.

Cell-free DNA (cfDNA) has become a promising noninvasive clinical marker widely studied in early disease detection, monitoring, and therapy selection. However, there is lack of data on a number of cfDNA-associated procedural features such as blood plasma biobanking conditions, isolation, and quantification methods that should be taken into account as they can affect downstream applications. In this study cfDNA from 125 plasma samples from healthy individuals were isolated using three different commercial kits (bead and vacuum based). Yield of cfDNA, distribution of cfDNA fragments and absolute amount of miR-223 were estimated. Moreover, the impact of different plasma biobanking conditions and hemolytic plasma were evaluated. In conclusion, results showed that quantification method (fluorescence or microcapillary electrophoresis based) has a major impact in estimating cfDNA amount. Samples isolated by QIAamp showed a higher amount of larger (around 300 bp) DNA fragments and miRNA yield, suggesting possible applications for multiomics approach. On the other hand, the highest cfDNA yield was obtained in samples isolated by the MagMAX Isolation Kit. This kit also showed lowest coefficient of variation and low miRNA yield. Plasma storage conditions and hemolysis affected performance of isolation kits differently.