MitoQuicLy: A high-throughput method for quantifying cell-free DNA from human plasma, serum, and saliva.

• Cell-free mitochondrial DNA (cf-mtDNA) is an emerging biomarker of psychobiological stress. • MitoQuicLy was developed to perform high-throughput quantification of cf-mtDNA in clinically relevant biofluids. • Within a person, cf-mtDNA levels vary up to ∼100-fold between biofluid types. • Correlations between cf-mtDNA and clinical biomarkers is biofluid dependent. • These results raise critical questions around the biological nature of cf-mtDNA in different biofluids. Circulating cell-free mitochondrial DNA (cf-mtDNA) is an emerging biomarker of psychobiological stress and disease which predicts mortality and is associated with various disease states. To evaluate the contribution of cf-mtDNA to health and disease states, standardized high-throughput procedures are needed to quantify cf-mtDNA in relevant biofluids. Here, we describe MitoQuicLy: Mito chondrial DNA Qu antification i n c ell-free samples by Ly sis. We demonstrate high agreement between MitoQuicLy and the commonly used column-based method, although MitoQuicLy is faster, cheaper, and requires a smaller input sample volume. Using 10 µL of input volume with MitoQuicLy, we quantify cf-mtDNA levels from three commonly used plasma tube types, two serum tube types, and saliva. We detect, as expected, significant inter-individual differences in cf-mtDNA across different biofluids. However, cf-mtDNA levels between concurrently collected plasma, serum, and saliva from the same individual differ on average by up to two orders of magnitude and are poorly correlated with one another, pointing to different cf-mtDNA biology or regulation between commonly used biofluids in clinical and research settings. Moreover, in a small sample of healthy women and men (n = 34), we show that blood and saliva cf-mtDNAs correlate with clinical biomarkers differently depending on the sample used. The biological divergences revealed between biofluids, together with the lysis-based, cost-effective, and scalable MitoQuicLy protocol for biofluid cf-mtDNA quantification, provide a foundation to examine the biological origin and significance of cf-mtDNA to human health. [ABSTRACT FROM AUTHOR]