Optimization of cell-free DNA isolation and detection by real time quantitative PCR in plasma samples

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

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

SAIC

Cell-free DNAs (cfDNA) are short DNA fragments derived from cell death and NETosis. cfDNA is emerging as a promising biomarker for pregnancy disorders. We optimized a method to isolate and detect cfDNA from plasma. Plasma samples from 18 to 40-year-old pregnant (during first, second and third trimesters) and non-pregnant were obtained by venipuncture in EDTA tubes. They were centrifuged at high revolutions to remove cell debris and cfDNA was isolated from 400 µL supernatants using QIAmp DNA Blood Mini Kit (QIAGEN). Several elution volumes (Ve) were assayed: 25, 40, 50 and 60 µL. cfDNA was detected by real time quantitative PCR of total cfDNArepresentative genes: B-Actin and Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH). Different cfDNA volumes (5 and 10 µL) and dilutions (1/2, 1/4 and 1/8) in a 20 µL final volume were assessed. In addition, different amplification conditions were analyzed, i.e.: primer concentration (0.25 and 0.5 pmol/µL), and annealing temperature (Ta) (B-Actin: 50, 51, 52.5 °C; GAPDH: 58.5, 60 °C). Similar results were obtained in pregnant and non-pregnant women. For GAPDH gene, non-specific amplification products were detected in all assays, while for B-Actin gene, it depends on the conditions assayed. Among all Ve assayed, 50 µL yielded a specific B-Actin amplification. Using a Ta=51 °C and 0.5 pmol/µL sense-antisense-primer both specific and no-specific amplification products were detected. However, using 0.25 pmol/µL sense-antisense-primer and 5 µL cfDNA eluate, only specific amplification products were detected, with Ct values above 28 and low standard deviation between duplicates (difference