Qiasymphony robot

cfDNA was purified from 4 mL of plasma using a QIAsymphony robot (Qiagen, Hilden, Germany), using a DSP Virus/Pathogen Midi-kit (ref. No. 937,055) as previously described [13 (link)]. The purified cfDNA was eluted into 60 μL of AVE buffer.

DNA from the two cell lines was isolated using the QIAamp Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Circulating-free DNA was purified as follows: 15 ml blood was withdrawn from patients and collected in Vacutainer tubes (BD, Plymouth, UK). Plasma and serum were isolated by centrifugation twice at 2300 r.p.m. for 10 min. The supernatant (serum or plasma) was aliquoted and used immediately for cfDNA isolation or stored at −80 °C. Cell-free DNA was purified from serum and plasma for each patient (1.2 ml). In the rare instances that the volume of the serum and plasma sample obtained from a patient was between 1 and 1.2 ml, PBS up to 1.2 ml was added to the samples, which were then purified using the QIAsymphony robot (Qiagen) and the QIAsymphony DSPVirus/Pathogen Midi Kit, according to the manufacturer's instructions, and cfDNA was eluted in a final volume of 30 μl. Since correct preanalytical handling of blood specimens is crucial to maintain the sample informative, the process was standardised (in terms of blood collection, sample centrifugation and cfDNA extraction) in the Department of Public Health of the University of Naples Federico II, and all procedures were performed in-house by a nurse belonging to the laboratory staff.