SIMPLE, EFFICIENT AND LOW-COST METHODOLOGY FOR CELL FREE RNAs EVALUATION IN HUMAN PLASMA AND URINE SAMPLES

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

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

Cell-free ribonucleic acids (cfRNAs) analysis could be used as biomarkers for monitoring different pathological conditions such as high-risk pregnancies. Most studies use commercial columns for high-quality isolation, ready-to-use RNA. However, it is important to have an efficient, simple, reliable and low-cost method. We optimized an alternative method for cfRNAs isolation using TRIzol reagent. Plasma and urine samples from 18 to 40-year-old women were used. Some of them were obtained from pregnant women (during the first, second and third trimester) and others from non-pregnant women. Plasma samples were obtained by venipuncture in ethylene diamine tetraacetic acid (EDTA) tubes. All samples were centrifuged at high revolutions to remove cell debris and cfRNAs were isolated from supernatants using TRIzol reagent (Invitrogen). Several volumes were assayed: 150, 250, 400 and 600 μL. Then, cfRNAs were converted to cDNA by retrotranscription (RT) using Moloney murine leukemia virus reverse transcriptase (Promega). The ribosomal protein L19 (L19) cfRNA was analyzed by real time quantitative PCR. Several cDNA dilutions were assayed: 1/2, 1/4 and 1/8. Amplification products were analyzed by 1.5% agarose gel electrophoresis. cfRNAs were purified in all volumes assayed, except in 150 μL plasma samples due to aqueous phase absence. cfRNAs concentrations were from 10.9 to 158.5 ng/μL. RT was carried out using 0.2-1 μg RNA. The L19 target was amplified in all samples using 5 μL cDNA. Furthermore, Cts values obtained demonstrated amplification progression in cDNA successive dilutions assayed, with low standard deviation between duplicates (difference