Minelute virus kit

DNA was extracted from tissue specimens and 800 ul of plasma using the QIAamp DNA Tissue Kit and MinElute Virus Kit (Qiagen, Valencia, CA, USA), respectively, according to the manufacturer’s recommendations. For plasma isolation, whole blood was centrifuged at 2600 rpm for 10 min at 10 °C. The collected material was centrifuged once again in 2-ml low-bind tubes at 14,500 rpm for 10 min to remove residual cells. All samples were stored at − 20 °C until cfDNA isolation. DNA quality and quantity were confirmed using a NanoDrop 1000 Spectrophotometer (Thermo Scientific) and Qubit Fluorometer (Thermo Scientific), respectively.

All plasma samples for this study were obtained from the Biobank of Taipei Veterans General Hospital. Cell-free DNAs from plasma were extracted using the QIAamp DNA Tissue Kit and Minelute Virus Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s recommendations. The quality and quantity of DNA were measured by using the Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
The quantity of cfDNA was represented by the copy number of the housekeeping gene cyclophilin (gCYC), a gene not known to be involved in cancer [30 (link)]. TaqMan qPCR assay (Thermo Fisher Scientific) was used to quantify the DNA copy number. Quantitative PCR was performed using TaKaRa Ex Master Mix. (Takara Bio, Shiga, Japan) Serially diluted standard DNAs were used to create a standard curve. Results were expressed as value of the threshold cycle (C_t), i.e., the cycle number at which the PCR product reached the threshold of detection. The final C_t values were normalized based on the standard curve. The gCYC qPCR results were used to normalize plasma sample DNA to the number of DNA alleles per mL.

DNA extraction from tissue specimens was performed using the QIAamp DNA Tissue Kit and MinElute Virus Kit (Qiagen, Valencia, CA) according to a previous report [8 (link)].