Nanodrop one microvolume spectrophotometer

Quantitative real-time polymerase chain reaction (qRT-PCR) is a useful technique for quantitative analysis of gene expression [38 (link)]. RNA extraction kits (Tian Gen, Beijing, China) were used to extract total RNA. After determination of the RNA concentration with a NanoDrop One Microvolume Spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA), and HiScript II Q RT SuperMix reagent (Vazyme, Nanjing, China) was added to all RNA samples (quantified to 1000 ng) for reverse transcription. The reverse-transcribed samples were cyclically amplified with ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) in a Roche LightCycler 480 (Roche, Basel, Switzerland) with GAPDH as the internal reference gene. The PCR conditions were as follows: 95°C for 30 s followed by 40 cycles at 95°C for 10 s and 60°C for 30 s. Data were processed using the 2^−ΔΔct calculation method. Primers were purchased from Sangon Biotech (Shanghai, China); detailed information regarding the primers is presented in Table 2.

DNA was isolated from thawed mammalian cell culture medium supernatant through automated nucleic acid extraction using the QIAsymphony^® (Qiagen). Fifty microliters elutes from the same culture medium were pooled and subsequently quantified using the Qubit™ dsDNA HS kit (Invitrogen, Thermo Fisher Scientific) on a Qubit 4 Fluorometer (Invitrogen, Thermo Fisher Scientific). Isolates were stored at −20°C. Mock cfDNA samples derived from cell culture were separated on a 1.5% UltraPure™ agarose gel (Invitrogen, Thermo Fisher Scientific) with 0.05% (v/v) EtBr and visualized with a Gel Doc XR+ imager (Bio-Rad Laboratories, Inc.) to confirm presence of expected nucleosomal fragment sizes.
For the tumor fraction simulation assay, mock cfDNA from VU1131 was diluted with mock cfDNA obtained from a TP53 wild-type cell line (WM9). Tissue biopsies were subjected to macrodissection before DNA isolation, ensuring that neoplastic cellularity was at least 20%. Subsequent DNA isolation was done using the Purelink™ Genomic DNA Mini Kit (Cat. No. K182001; Invitrogen, Thermo Fisher Scientific). Nucleic acid concentration and purity were assessed using a NanoDrop One microvolume Spectrophotometer (Thermo Fisher Scientific).

Viral DNA extracted from the frozen supernatant was prepared using a commercial kit according to the manufacturer’s instructions (PureLink Viral RNA/DNA Mini Kit, Invitrogen, Carlsbad, CA, USA). DNA purity and concentration were assessed using a NanoDrop One Microvolume Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Samples were confirmed to contain FHV-1 DNA using quantitative polymerase chain reaction (qPCR; ABI 7900-2; Waltham, MA, USA) on extracted DNA using thymidine kinase primers, which are specific for FHV-1 (IDT, Coralville, IA, USA).