153 Detection of non-metastatic non-small cell lung cancer in urine and smoking history were documented. URIC participants were selected on having the same age range as the NSCLC cases. The Medical Ethical Committee board of the Amsterdam UMC approved the study design including the collection of urine from NSCLC patients (no. 2017.333 and no. 2017.545) and healthy volunteers (no. 2017.112). Written informed consent was obtained from all participants of this study. Urine collection and processing Preoperative urine samples were collected autonomously by participants at home at least two weeks before planned pulmonary surgery. Ambulant urine collection was realized by providing participants a collection kit which included a large collection container (300 ml) and three 30 ml collection tubes. The collection tubes contained 2 ml of 0.6 M Ethylenediaminetetraacetic acid (EDTA) as a preservative agent (final concentration of 40 mM). Study participants sent their urine samples to the Department of Pathology of Amsterdam UMC, location VUmc by mail, where samples were processed within 24-72 h, following collection. This collection and storage protocol was previously validated (26). Postoperative urine samples were usually collected at outpatient clinic visits, or by autonomous collection at home as described above. To acquire the urine supernatant fraction, samples were centrifuged at 3000g for 15 min. All urine samples were stored at -20 °C until DNA isolation. DNA isolation and bisulfite modification DNA was isolated from 20 ml urine supernatant using the Quick DNA urine kit (Zymo Research, Irvine, CA, US). Extracted DNA was eluted in 50 μl elution buffer, after which DNA concentrations were measured using the Qubit™ dsDNA HS Assay (Invitrogen, Carlsbad, CA, US). To allow for DNA methylation analysis, up to 250 ng of isolated DNA was subjected to sodium bisulfite treatment using the EZ DNA Methylation kit (Zymo Research, Irvine, CA, US). All procedures were performed according to the manufacturer’s guidelines. DNA methylation analysis DNA methylation analysis was performed using quantitative methylation-specific PCR (qMSP), as described previously (25). Briefly, a multiplex qMSP targeting the hypermethylated promoter regions of 3 genes (CDO1, SOX17, and TAC1) and a reference gene (ACTB) was developed based on gene loci discovered in Hulbert et al. (17) and adjusted for NSCLC detection in urine in Liu et al. (19). Amplicon sizes did not exceed 70 base pairs, facilitating the detection of methylation in small DNA fragments present in urine. The qMSP analysis was performed on a ViiA7 real-time PCR-system (Applied 6
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