OPTIMIZATION OF CELL-FREE DNA ISOLATION AND DETECTION BY REAL TIME QUANTITATIVE PCR IN PLASMA SAMPLES.

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

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

Cell-free DNAs (cfDNA) are short DNA fragments derived from cell death and NETosis. cfDNA isemerging as a promising biomarker for pregnancy disorders. We optimized a method to isolateand 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 from400 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 totalcfDNA representative 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 μLfinal 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-pregnantwomen. 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 μLyielded a specific B-Actin amplification. Using a Ta=51 °C and 0.5 pmol/μL sense-antisenseprimerboth specific and no-specific amplification products were detected. However, using0.25 pmol/μL sense-antisense-primer and 5 μL cfDNA eluate, only specific amplificationproducts were detected, with Ct values above 28 and low standard deviation betweenduplicates (difference