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

Abstract

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: Mitochondrial DNA Quantification in cell-free samples by Lysis. 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.

Keywords: Cell-free DNA; Circulating nucleic acids; DNA isolation; Mitochondria; Mitochondrial DNA; Protocol.

Figure 1:. Optimizing collection, lysis and qPCR protocol.

(A) Schematic of MitoQuicLy Protocol. Biological samples are collected (min 5 mL) and centrifuged twice to remove cells. Cell-free samples are added to lysis buffer in a 96-well plate, sealed with cap strips and incubated overnight in a standard thermocycler before quantification using qPCR. (B) Schematic of lysis reaction. Tween-20 solubilizes membranes and Proteinase K digests proteins. The lysate is used directly as template DNA for qPCR. (C) Pooled EDTA plasma was lysed at a range of concentrations. At higher concentrations (≥5%), a stringy white precipitate forms (blue arrows). (D) DNA was quantified from either the supernatant (grey) or the precipitate (blue) from lysates at 10% plasma (plasma diluted 1:10 with lysis buffer), indicating the high abundance of DNA in the precipitate. No nDNA was detected in supernatants (Ct undetermined: >40), but nDNA was detected in precipitates albeit at a high Ct. N.D.: Not detected. ****p<0.0001, unpaired t test. (E) Samples with precipitation have less DNA than unprecipitated samples. Plasma was lysed in a dilution series from 10% to 0.5%. A linear relationship between cycle threshold and plasma concentration was observed below 2.5%, but not above 2.5%; simple linear regression. (F) Dilution series from 10 participants lysed at a series of concentrations from 10% to 0.5% (inset) and at concentrations from 2.5% to 0.05%, confirming the assay breakpoint, and linearity below 2%. Each participant datapoint was measured in triplicates.

Figure 2:. Modifying buffer component concentrations.

(A) A range of buffer compositions were tested (see supplement) to compare low (L) and high (H) buffer component, yielding 5 different buffer combinations tested against the original LLL (using the modal maximum values in single buffer experiments). (B and C) Citrate plasma and saliva cf-mtDNA levels measured in matching sample types from n=6 individuals, indicating improved yield with revised buffer combinations. Each datapoint was measured in triplicates. (D) mtDNA copies detected for each buffer combination relative to LLL. (E and F) Fold-change in cf-mtDNA yield relative to the LLL buffer, revealing HHL as optimal for citrate plasma and saliva. n=6 individuals, 3 women, 3 men; p<0.05*, p<0.01**, p<0.001***, p<0.0001****, two-way ANOVA.

Figure 3:. Titration of sample concentration with optimized MitoQuicLy buffer.

(A and E) ND1 quantification in citrate plasma and saliva from n=10 people lysed in eight 2-fold serial dilution ranging from 20% to 0.078% (vol/vol). Each person’s serial dilution is represented by points on a connected line. (B and F) Copies/μL derived from a standard curve, showing that the assay in citrate plasma is linear from 0 to 10% plasma, but “breaks” between 10 and 20% (grey shading). In saliva, the assay range is linear across all concentrations tested. (C and G) Dilution-corrected copies/μL in plasma and saliva. From 0–10% plasma or saliva, all samples are reliably rank-ordered at any of the titrated concentrations. (D and H) Spearman’s r for plasma and saliva dilution curves (copies/μL) illustrating high correlations between all dilution ≤10%. n=10 individuals, 5 women, 5 men.

Figure 4:. MitoQuicLy variability.

(A) cf-mtDNA quantified on pooled samples from 34 healthy controls (14 women, 20 men) were run as 96 technical replicates using MitoQuicLy. Frequency distributions of cycle thresholds (Ct, left) or copies/μL (right) for citrate plasma (B), EDTA plasma (C), serum (D), and saliva (G). CVs were acceptable for all samples except EDTA plasma, likely due to the low DNA concentration in EDTA plasma.

Figure 5:. Comparison of cf-mtDNA levels between MitoQuicLy and silica-based membrane kit.

(A) cf-DNA quantified from various plasma or serum types using either MitoQuicLy or a silica-based membrane column DNA extraction kits (DNeasy, Qiagen). A total of 50 samples were analyzed (5 biofluids, n=10 individuals [5 women, 5 men]). (B) Data from (A) color-coded by sample type. (C) mtDNA and (D) nDNA variability via column method. CVs are shown below the x-axis. 8 replicates per sample type. (E) mtDNA and (F) nDNA abundance from the same sample as measured by the column method or MitoQuicLy. n.d.: not detected. (G) mtDNA and (H) nDNA percent recovery by method (relative to the column method). Heparin plasma was excluded from the nDNA comparison, as MitoQuicLy did not detect heparin plasma nDNA. (I) Procedural and financial comparison of key parameters between MitoQuicLy and standard silica-based membrane DNA extraction kits. Blue cells indicate advantages of MitoQuicLy, grey cells indicate advantages of the column method. n=5–6 sample types, **p<0.01, paired t-tests.

Figure 6:. MitoQuicLy reveals sample type differences in cf-mtDNA.

(A) Schematic of experimental design for MitoQuicLy cf-mtDNA analysis across 6 biofluids. (B) cf-mtDNA cycle threshold by sample type, revealing differences in cf-mtDNA levels between sample types, and substantial inter-individual differences. Each datapoint represents a participant (n=34 per biofluid). (C) Fold differences in cf-mtDNA copies/μL normalized to EDTA plasma cf-mtDNA copies/μL for each individual (each participant set to 1 for EDTA, connected by a line across sample types). The average fold difference for each sample type across the cohort is indicated. (D) Spearman’s r of cf-mtDNA copies/μL between sample types. Only red-top and gold-top serum cf-mtDNA levels show a significant correlation with each other. Plasma cf-mtDNA levels are not significantly correlated with serum or saliva, and saliva cf-mtDNA levels are not significantly correlated with any sample type. (E) Sex differences in cf-mtDNA levels by sample type. Shown are Hedges’ g (standardized effect size) and P values from unpaired t-tests, n=34 participants (14 women, 20 men).

Figure 7:. MitoQuicLy reveals different biological associations with cf-mtDNA by sample type.

(A) Experimental design to combine MitoQuicLy cf-mtDNA levels from 6 biofluids and a standard blood biomarker panel. (B) Heatmap of correlations (Spearman’s r) between cf-mtDNA levels measured in each biofluid (n=34 individuals per sample type) and blood biomarkers. P values are not corrected for multiple comparisons. (C-E) Selected correlations (Spearman r) between blood biomarkers and cf-mtDNA levels. Each datapoint represents a participant (n=34; 14 women, 20 men). Spearman’s r correlations and p-values not corrected for multiple testing; p<0.05*, p<0.01**, p<0.001***, p<0.0001****. Abbreviations: BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Hgb A1C, hemoglobin a1c; RBC, red blood cell count; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red cell distribution width; INR, international normalized ratio of prothrombin time; APTT, activated partial thromboplastin time; PLT, platelet count; MPV, mean platelet volume; WBC, white blood cell count; BUN, blood urea nitrogen; CRP, c-reactive protein.