Nf h2o

Due to NucleoSnap cfDNA kit’s (Macherey-Nagel, Düren, Germany) superior performance in the first comparative experiments, we directly compared NucleoSnap to the NucleoSpin miRNA Plasma kit (Macherey-Nagel, Düren, Germany) concerning cfDNA yield and purity. Moreover, we evaluated cfRNA yields from both kits. For NucleoSpin, all samples were centrifuged at 4500× g before the start of the protocol. Skipping the optional DNA digestion step allowed for the parallel extraction of cfDNA and cfRNA. The final elution volume was 50 µL nuclease-free water (nf-H₂O; Thermo Fisher Scientific, Waltham, MA, USA) after 5 min incubation on the membrane. Co-isolated cfNA was stored at −80 °C until further analysis.

DNA samples that showed a high degree of gDNA contamination in BA profiles were subjected to size selection with AMPure XP beads (Beckman Coulter, Brea, CA, USA). Size selection was performed as previously described [60 (link),61 (link)]. AMPure XP beads (Beckman Coulter, Brea, CA, USA) were added to the sample in a 0.6× ratio, incubated for 5 min at RT, and then put onto a magnet for 3 min. The supernatant was transferred into a new 1.5 mL Eppendorf tube (Eppendorf, Hamburg, Germany) and new beads were added to a 2.0× ratio. Samples were incubated for 5 min at RT and put onto a magnetic rack for 3 min. The supernatant was discarded. The beads were washed with fresh 80% ethanol (VWR International, Radnor, PA, USA) three times, followed by elution in 30 µL nuclease-free water (nf-H₂O, Thermo Fisher Scientific, Waltham, MA, USA). After 5 min incubation time at RT, the tubes were transferred to a magnetic rack and incubated for 3 min. The supernatant was transferred into a fresh tube (2.0× sample). Samples were stored at −80 °C until further processing. (SOP, cf. Protocol A4).