36 Chapter 2 correlation. Correlation coefficient r was defined as moderate (r = 0.40 – 0.59), strong (r = 0.60 – 0.79), or very strong (r = 0.80 – 1.00). Differences in DNA methylation levels amongst each urine fraction (i.e. full void urine, urine sediment, and urine supernatant), and between patients and controls were evaluated by comparing the log2-transformed Cq ratios. Cq ratios were computed by normalizing the methylation levels of all markers according to the reference gene ACTB using the comparative Cq method (2−ΔCq x 100). Methylation levels of all urine fractions of both patients and controls were displayed in boxplots and tested for statistical significance using the non-parametric Mann-Whitney U test. The diagnostic potential of GHSR, SST, and ZIC1 for distinguishing patients and controls were evaluated by computing receiver operating characteristic (ROC) curves of all methylation markers, and results were quantified by the area under the curve (AUC). Statistical analysis was performed in IBM SPSS 26, and graphs were created using GraphPad Prism 8. RESULTS Patient characteristics A total of 42 EC patients and 46 healthy controls were enrolled in this study. An overview of clinical characteristics is displayed in Table 1. DNA quality of urine fractions To select the most suitable urine fraction for DNA methylation analysis, the quality of DNA isolated from paired full void urine, urine sediment, and urine supernatant samples was first assessed by comparing the quantification cycle (Cq) values of the reference gene ACTB (Table 2). While the Cq values of ACTB were nearly identical in full void urine samples (24.7) and urine sediments (24.8), they were significantly higher (p < 0.001) in urine supernatant samples (26.1). Of note, amongst the different fractions, none of the samples tested invalid in urine sediment, as compared to two in both full void urine and urine supernatant samples.
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