35 Non-invasive detection of endometrial cancer by DNA methylation analysis in urine Urine collection and processing Both patients and controls collected urine at home in three 30 mL collection tubes, containing 2 mL 0.6 M Ethylenediaminetetraacetic acid (EDTA) as a preservative agent (final concentration of 40 mM). Urine samples were shipped to the Pathology department of Amsterdam UMC, VU University Medical Center, by regular mail and processed within 24 – 72 hours after collection. 15 mL of full void urine was centrifuged at 3000 x g for 15 minutes to separate the urine sample into two fractions: the sediment and the supernatant. The urine sediment, urine supernatant, and remaining full void urine were stored at -20 °C. This collection and storage protocol has previously been validated for reliable DNA methylation detection in urine (36). DNA extraction and bisulfite modification DNA was extracted and modified from full void urine, urine sediment, and urine supernatant as described before (22, 23). Briefly, DNA was isolated from full void urine (15 mL) and urine supernatant (15 mL) using the Quick DNA urine kit (Zymo Research, Irvine, CA, US). DNA was isolated from the urine sediment (15 mL original volume) using the DNA mini and blood mini kit (Qiagen, Hilden, Germany). DNA concentration and DNA quality were measured using a NanoDrop 1000 (ThermoFisher Scientific, Waltham, MA, US). 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 guidelines. DNA methylation analysis by quantitative methylation-specific PCR (qMSP) DNA methylation analysis of GHSR, SST, and ZIC1 was executed by multiplex qMSP, including ACTB, using 50 ng modified DNA input on an ABI-7500 real-time PCR-system (Applied Biosystems, Waltham, MA, US), as described previously (22, 37). ACTB was used as a reference gene for quantification and quality assessment. Sample quality was ensured by excluding samples with a quantification cycle (Cq) value exceeding 32 from methylation analysis. Data analysis The DNA quality of each urine fraction of both patients and controls, of which all paired fractions were available, was examined by comparing their median ACTB Cq values using the Friedman Test, followed by the non-parametric Wilcoxon signed-rank test. In addition, the number of samples tested invalid (i.e. excluded due to an ACTB Cq value ≥ 32) was documented per urine fraction. The correlation between Cq ratios of each DNA methylation marker between paired urine fractions of both patients and controls was assessed using Spearman’s rank 2
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