Cell free DNA isolation and detection in human urine

CEPEDA PJ; RACCA ME; MILESI MM; VARAYOUD J; MUÑOZ-DE-TORO M; ROSSETTI MF; RAMOS JG

Congreso; LXVI REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC); 2021

SAIC

Cell free DNA (cfDNA) exists in different biological fluids and is proposed as a potential diagnostic indicator for different clinical conditions. Urine CfDNA detection would provide a tool for a less invasive prospective diagnosis. Limited information about urinary cfDNA detection is available, and cfDNA stability is a challenge due to urine variable pH. We optimized a reliable method for cfDNA isolation and detection from urine. Urine samples from 18 to 40-year-old women were centrifuged at high revolutions to remove cell debris. To assess whether urine acidity influences isolation efficiency, acidic (pH5) and neutralized (pH7) urine samples were assayed. cfDNA was isolatedfrom 800 µL urine samples aliquots using QIAamp DNA Blood Mini Kit (QIAGEN) and eluted with 50 µL elution buffer. cfDNA concentration was measured by Nanodrop spectrophotometer. A real timequantitative PCR was performed using an optimized protocol for B-Actin amplification. Different cfDNA volumes (5 and 10 µL) and dilutions (1/2, 1/4 and 1/8) in a 20 µL final volume were assayed.Amplification products were analyzed by 1.5% agarose gel electrophoresis. cfDNA concentrations in neutralized and non-neutralized samples were 4.7 and 5.3 ng/µL, respectively. Specific B-Actin amplification product, in both neutralized and non-neutralized samples, was detected at Tm=80.1 °C; but at Tm=75.4 °C a non-specific amplification product was also detected. Sample neutralization prior to isolation considerably decreased non-specific amplification products. Furthermore, Cts values obtained demonstrated amplification progression in cfDNA successive dilutions assayed, with low standard deviation between duplicates (difference