Quantification of cell-free DNA isolated from cell culture supernatant: Method comparison

Recently, the aims of many cell-free DNA (cfDNA) studies have been directed towards gaining insight of its biological properties, in pursuit of being able to develop and implement cfDNA-based tests in clinical environments. The characterization of cfDNA in vivo has proven to be increasingly problematic due to the inherently complex composition of blood. Therefore, the use of less complex and thus more manageable models, such as in vitro cell cultures, have gained some popularity. Regardless of the advantages of in vitro solutions, three important limitations should be addressed. These include: i) cfDNA concentrations in vitro are low; ii) the amounts and sizes of in vitro cfDNA fluctuates notably over time; iii) different cell types exhibit significantly different cfDNA amounts and sizes. Without method standardization, these points are causes of considerable variability, and can lead to inconsistencies and disagreements within the research field. Therefore, as a step towards the standardization of in vitro cfDNA experiments, we used cell culture supernatants collected from HMEC-1 cells at different time intervals (to simulate different cfDNA concentrations) to compare the performance of five cfDNA quantification methods (qPCR, Bioanalyzer HS DNA assay, Quant-iT PicoGreen Assay, Qubit dsDNA HS assay, and NanoDrop One^c). We determined that even though all methods were capable of detecting cfDNA with good reproducibility, the different methods measured significantly different cfDNA concentrations (ANOVA p < 0.0001). When methods were compared to qPCR, the current gold standard in DNA quantification, cfDNA concentrations were overestimated by all four the other methods by the following percentages: NanoDrop One^c (247.6 ± 12.2 %), Bioanalyzer HS DNA kit (114.8 ± 16.3 %), Quant-iT PicoGreen Assay (59.8 ± 9.9 %), and Qubit dsDNA HS assay (22.4 ± 6.7 %). Thus, the Qubit delivered results comparable to qPCR, and could therefore be considered a viable alternative for cfDNA quantification.