Annals of clinical biochemistry · 2009
A rapid direct fluorescent assay for cell-free DNA quantification in biological fluids.
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Goldshtein Hagit, Hausmann Michael J, Douvdevani Amos
Аннотация
BACKGROUND: 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.
METHODS: 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).
RESULTS: 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).
CONCLUSIONS: The direct SYBR Gold assay proved to be an accurate and simple technique for measuring CFD in biological fluids.
METHODS: 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).
RESULTS: 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).
CONCLUSIONS: The direct SYBR Gold assay proved to be an accurate and simple technique for measuring CFD in biological fluids.