A rapid direct fluorescent assay for cell-free DNA quantification in biological fluids

Abstract

Circulating cell-free DNA (CFD) levels may be elevated in trauma, stroke, sepsis, pre-eclampsia and cancer. Owing to the complex and expensive methodology, detection of CFD has hitherto been confined to research laboratories. This study presents a simple, inexpensive and accurate test for CFD. Using the commercial fluorescent SYBR Gold stain, biological fluids were directly assayed for CFD without prior DNA extraction and amplification. Stain was added to the sample in 96-well plates (final stain dilution: 1:10,000) and fluorescence was read by a fluorometer (excitation wavelength 488 nm, emission wavelength 535 nm). The assay was validated with serum, whole blood, urine and supernatant of cell cultures. Specificity and linearity were demonstrated over a wide range of concentrations; the results correlated with the conventional quantitative polymerase chain reaction assay of beta-globin (R(2) = 0.9987, P < 0.001). The assay was not affected by exposure of whole blood or serum to room temperature for four or 24 h, respectively. Intra and day-to-day coefficients of variation (16-4.8% and 31-8%, respectively; depending on DNA level) compared well with published data describing more work-intensive tests. The limit of quantitation (170 ng/mL) was below the mean DNA level in a cohort of normal individuals (471 [203] ng/mL). Finally, free DNA in supernatant of cell cultures after cell lysis accurately reflected cell number (R(2) = 0.974, P < 0.0001). The direct SYBR Gold assay proved to be an accurate and simple technique for measuring CFD in biological fluids.