Cell free DNA isolation and detection in human urine

CEPEDA, P.J.; VARAYOUD, J; RAMOS, J.G; RACCA, M.E.; MUÑOZ-DE-TORO, M; MILESI, M.M; ROSSETTI, M.F

Virtual

Congreso; Reunión Conjunta SAIC. SAI. AAFE. NANOMED-AR 2021; 2021

Cell free DNA (cfDNA) exists in different biological fluids and is proposed as a potentialdiagnostic indicator for different clinical conditions. Urine CfDNA detection would provide atool for a less invasive prospective diagnosis. Limited information about urinary cfDNAdetection is available, and cfDNA stability is a challenge due to urine variable pH. Weoptimized a reliable method for cfDNA isolation and detection from urine. Urine samples from18 to 40-year-old women were centrifuged at high revolutions to remove cell debris. To assesswhether urine acidity influences isolation efficiency, acidic (pH5) and neutralized (pH7) urinesamples were assayed. cfDNA was isolated from 800 µL urine samples aliquots using QIAampDNA Blood Mini Kit (QIAGEN) and eluted with 50 µL elution buffer. cfDNA concentration wasmeasured by Nanodrop spectrophotometer. A real time quantitative PCR was performed usingan optimized protocol for B-Actin amplification. Different cfDNA volumes (5 and 10 µL) anddilutions (1/2, 1/4 and 1/8) in a 20 µL final volume were assayed. Amplification products wereanalyzed by 1.5% agarose gel electrophoresis. cfDNA concentrations in neutralized and nonneutralized samples were 4.7 and 5.3 ng/µL, respectively. Specific B-Actin amplificationproduct, in both neutralized and non-neutralized samples, was detected at Tm=80.1 °C; but atTm=75.4 °C a non-specific amplification product was also detected. Sample neutralization priorto isolation considerably decreased non-specific amplification products. Furthermore, Ctsvalues obtained demonstrated amplification progression in cfDNA successive dilutionsassayed, with low standard deviation between duplicates (difference