179 Dynamics of methylated cell-free DNA in the urine of non-small cell lung cancer patients Urine samples of patients and controls were processed similarly. Up to 30 mL (patients) or 40 mL (controls) full void urine was centrifuged at 3000 × g for 15 min to obtain the urine supernatant fraction, which was stored at − 20 °C. This collection and storage procedure has been validated for reliable DNA methylation detection in urine (18). Cell-free DNA extraction and bisulfite conversion 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. DNA methylation analysis by quantitative methylation-specific PCR (qMSP) Promoter hypermethylation detection of the CDO1, SOX17, and TAC1 genes was carried out by qMSP using a ViiA7 real-time PCR system (Applied Biosystems, Foster City, CA, USA). For each reaction, up to 50 ng modified DNA was mixed with the EpiTect MethyLight Master Mix (Qiagen), and 2.5-5.0 µM of each primer and 5.0-10.0 µM of each probe in a total volume of 12.5 µl. Primer and probe sequences used for CDO1 and TAC1 were kindly provided by Dr. A. Hulbert (University of Illinois at Chicago, Chicago, IL, US) and listed in (10). Primer and probe sequences of SOX17 were redesigned within the same genomic region as reported before (10), using a locked nucleic acid probe to enhance specificity (Supplementary Table 1). The qMSP reactions were multiplexed as described previously (27) to assess the methylation levels of all genes within the same reaction. ACTB was also included in the multiplex and used as a reference gene for normalization and quality assessment. Sample series from each patient were processed in the same run. Double-stranded gBlocks™ Gene Fragments (Integrated DNA Technologies) containing the amplicon sequences of all targets and ACTB were used as technical quality control and H2O was taken along as negative control during each qMSP run. Cycle threshold (Ct) values were measured at a fixed threshold. Sample quality and sufficient input were ensured by excluding samples with a ACTB Ct value exceeding 32 from methylation analysis. The discriminatory power of the qMSP was verified by testing 11 pairs of tumors and adjacent normal tissues from NSCLC patients of a previously published cohort (28, 29). 7
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