Quick dna urine kit

Approximately 50 ml of urine was processed per patient, with 10 ml of urinary supernatant or the entire pellet utilised for DNA extraction using the Quick-DNA Urine kit (Zymo, D3061). DNA concentrations were determined using the Qubit high sensitivity DNA kit (Thermo, #Q32854) and cfDNA size determined by Bioanalyzer using high sensitivity chips (Agilent, # 5067-4626). cfDNA preparations from 104 patients yielded >10 ng DNA and were included for analysis. The overall study design and patient demographics are shown in Supplemental Figure S1 and Table S1. The number of wt and TERT 228 G>A/T DNA molecules was determined in all urinary DNAs and 21 of the tumour DNAs by digital droplet PCR using the TERT_C228T liquid biopsy assay (Thermo, #A44177), supermix for probes (Biorad, #1863010) and the Biorad QX200 droplet formation/reader system (Biorad). The PCR program consisted of 10 minutes at 96°C followed by 54 cycles of 98°C×30 sec and 55°C×2 min. Log rank tests were used to compare data across patient groups.

The urinary cfDNA was extracted from 20 mL (patients) or 40 mL (controls) urine supernatant using the Quick DNA urine kit (Zymo Research, Irvine, CA, US). Previous research showed that differences in urine collection volume in a similar range (4–20 mL) have limited effects on DNA yield, eliminating this potential bias [26 ]. DNA concentration was measured using the Qubit™ dsDNA HS Assay (Invitrogen, Carlsbad, CA, US). Depending on the yield, up to 250 ng purified DNA was subjected to bisulfite conversion using the EZ DNA Methylation Kit (Zymo Research). All procedures were carried out according to the manufacturer’s instructions.