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; Reunión Conjunta de Sociedades de Biociencias 2021: SAIC, SAI, AAFE, NANOMED-ar.; 2021

Cell-free DNA (cfDNA) originates mainly from cell death and NETosis. Itcan cross the kidney barrier and be excreted in urine, as well as, be generatedfrom urinary tract cell turnover. Limited information about urine cfDNAdetection is available. Urine is a non-invasive sample and large volumes areavailable, however, urine-cfDNA stability is challenging due to its variablepH. We optimized a cfDNA isolation and detection method from urine samples.Urine samples from 18 to 40-year-old women were centrifuged at high revolutionsto remove cell debris. To assess whether urine acidity influences isolation efficiency,acidic (pH5) and neutralized (pH7) urine samples were assayed. cfDNA wasisolated from 800 µL urine samples aliquots using QIAamp DNA Blood Mini Kit(QIAGEN) and eluted with 50 µL elution buffer. cfDNA concentration was measuredby Nanodrop spectrophotometer. A real time quantitative PCR was performed usingan optimized protocol for B-Actinamplification. Different cfDNA volumes (5 and 10 µL) and dilutions (1/2, 1/4and 1/8) in a 20 µL final volume were assayed. Amplification products wereanalyzed by 1.5% agarose gel electrophoresis. cfDNA concentrations inneutralized and non-neutralized samples were 4.7 and 5.3 ng/µL, respectively.Melting temperature (Tm) demonstrated a specific B-Actin amplification product (Tm=80.1°C) in both neutralized andnon-neutralized samples, but a non-specific amplification product (Tm=75.4°C)was also detected. However, sample neutralization prior to isolationconsiderably decreased non-specific amplification products. Furthermore, Ctsvalues obtained demonstrated amplification progression in cfDNA successivedilutions assayed, with low standard deviation between duplicates (difference<0.5). We isolated and detected cfDNA from urine, and demonstrated theimportance of performing sample neutralization prior to isolation. Althoughlimited information about urine-cfDNA is available, it may be a promisingbiological biomarker.