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

This version was published on 1 November 2009

Ann Clin Biochem 2009;46:488-494
doi:10.1258/acb.2009.009002
© 2009 Association for Clinical Biochemistry

 

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Original Articles


Hagit Goldshtein1,
Michael J Hausmann2 and
Amos Douvdevani1,2


1 Department of Clinical Biochemistry, Ben-Gurion University of the Negev;
2 Department of Nephrology, Soroka Medical University Center, Beer-Sheva, Israel


Corresponding author: Amos Douvdevani, Nephrology Laboratory, Soroka Medical Center, Sderot Rager, P.O. Box 151, Beer-Sheva 84101, Israel. Email: amosd{at}bgu.ac.il


Background: Circulating cell-free DNA (CFD) levels may be elevated in trauma,stroke, sepsis, pre-eclampsia and cancer. Owing to the complexand expensive methodology, detection of CFD has hitherto beenconfined to research laboratories. This study presents a simple,inexpensive and accurate test for CFD.

Methods: Using the commercial fluorescent SYBR® Gold stain, biologicalfluids were directly assayed for CFD without prior DNA extractionand amplification. Stain was added to the sample in 96-wellplates (final stain dilution: 1:10,000) and fluorescence wasread by a fluorometer (excitation wavelength 488 nm, emissionwavelength 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 β-globin (R2 = 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 (R2 = 0.974, P < 0.0001).

Conclusions: The direct SYBR® Gold assay proved to be an accurate andsimple technique for measuring CFD in biological fluids.

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