Magmax cell free total nucleic acid isolation kit

Peripheral blood was collected into 10.0 mL BD Vacutainer^® plastic tubes containing EDTA (BD Diagnostics, Milan, Italy). The plasma fraction was obtained and stored as previously described within 2 h of the blood drawing [19 (link)]. Circulating cell-free total nucleic acid (cfTNA) was extracted from 4 mL of plasma with the MagMAX Cell-Free Total Nucleic Acid Isolation Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The circulating cell-free DNA (cfDNA) quantity was estimated using the Qubit 2.0 Fluorometer (Invitrogen, Waltham, MA, USA).

Saliva samples were collected as oral rinses. Patients were asked to swish 10 mL of 0.9% sodium chloride in their mouths for 10 to 15 s before spitting into the collection tube. Venous blood (4–10 mL) was collected using standard phlebotomy techniques in EDTA tubes and maintained at 4 °C until further processing. Plasma was obtained from blood within 4 h of phlebotomy upon two sequential centrifugations: the first at 1200× g for 7 min at 4 °C and the second at 14,000× g for 10 min at 4 °C. In some instances, the buffy coat layer was recovered during blood processing, lysed using a BD FACS Lysing Solution (Thermo Fisher Scientific, Waltham, MA, USA), centrifuged at 3000 rpm for 10 min, and stored at −80 °C. The saliva genomic DNA (gDNA) was extracted from 400 μL of oral rinses using a MagMAX Saliva gDNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA). Plasma-circulating cell-free DNA (cfDNA) was extracted from 4 mL of plasma using a MagMAX Cell-Free Total Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA). Leucocyte DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). DNA was quantified using Qubit Fluorometric Quantification (Thermo Fisher Scientific, Waltham, MA, USA).

Peripheral blood (1–10 ml) was collected in EDTA tubes and processed within a median of 1 h and 30 min by centrifugation at 2000 × g for 10 min to separate plasma and a buffy coat. The supernatant was removed, and a second centrifugation step was performed at 16,000 × g for 10 min to remove cell debris. All centrifugation steps were performed at 4 °C. The plasma was frozen until DNA isolation. cfDNA was extracted from 500 ul to 9 ml (median 3 ml) plasma samples using the MagMAX™ Cell-Free Total Nucleic Acid Isolation Kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA). The abundance and quality of cfDNA in the extracted samples were assessed using a bioanalyzer Cell-free DNA ScreenTape analysis (Agilent, Santa Clara, CA) and Quantus™ Fluorometer (Promega, Madison, WI).