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

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

Modalidad virtual

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

Cell-free ribonucleic acids (cfRNAs) analysis could be used as biomarkers for monitoringdifferent pathological conditions such as high-risk pregnancies. Most studies use commercialcolumns for high-quality isolation, ready-to-use RNA. However, it is important to have anefficient, simple, reliable and low-cost method. We optimized an alternative method forcfRNAs isolation using TRIzol reagent. Plasma and urine samples from 18 to 40-year-oldwomen 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 wereobtained by venipuncture in ethylene diamine tetraacetic acid (EDTA) tubes. All samples werecentrifuged at high revolutions to remove cell debris and cfRNAs were isolated fromsupernatants using TRIzol reagent (Invitrogen). Several volumes were assayed: 150, 250, 400and 600 µL. Then, cfRNAs were converted to cDNA by retrotranscription (RT) using Moloneymurine leukemia virus reverse transcriptase (Promega). The ribosomal protein L19 (L19) cfRNAwas analyzed by real time quantitative PCR. Several cDNA dilutions were assayed: 1/2, 1/4 and1/8. Amplification products were analyzed by 1.5% agarose gel electrophoresis. cfRNAs werepurified in all volumes assayed, except in 150 µL plasma samples due to aqueous phaseabsence. cfRNAs concentrations were from 10.9 to 158.5 ng/µL. RT was carried out using 0.2-1µg RNA. The L19 target was amplified in all samples using 5 µL cDNA. Furthermore, Cts valuesobtained demonstrated amplification progression in cDNA successive dilutions assayed, withlow standard deviation between duplicates (difference