URINE CELL-FREE DNA AS A POTENTIAL BIOMARKER FOR OBSTETRICS COMPLICATIONS. AN ALTERNATIVE DETECTION METHOD

RACCA ME; CEPEDA PJ; ROSSETTI MF; RAMOS JG; CARDOZO MA; LUQUE EH; MUÑOZ DE TORO, M; MILESI MM; VARAYOUD J

Congreso; Reunion Anual de Sociedades de Biociencias; 2022

Urine cell-free DNA (cfDNA) could serve as a biomarker for differentdiseases (e.g., obstetric complications). Noninvasive sampling andthe possibility to analyze large volume samples are advantages ofurine-based protocols. Urine cell-free DNA assessment usually includescommercial kits. We aimed to determine the efficiency of analternative purification method to extract and quantify urine cfDNA,comparing cfDNA levels on pregnant (P) and non-pregnant (NP)women. cfDNA was isolated from urine samples of P (first, secondand third trimester, n=47) and NP (n=23) women. After urine pHneutralization and high-revolution centrifugation, cfDNA was purifiedfrom 800 μL supernatant applying an organic extraction method.First, urine samples were lysed with 5M NaCl and 20% sodium dodecylsulfate solutions, then cfDNA was extracted with chloroformfollowed by ethanol precipitation. cfDNA was resuspended in 50 μLTris-EDTA buffer and resulting concentrations were measured byUV spectrophotometry. Purified cfDNA integrity was analyzed byagarose gel electrophoresis. Finally, cfDNA levels were quantifiedby real time PCR amplification of Actin-β gene (ACTB). cfDNA wasdetected in 69% urine samples and yielded specific ACTB amplificationwith low standard deviation between PCR duplicates (Ct difference