MS-HRM was performed on a LightCycler 480 machine (Roche Applied Science, Mannheim, Germany) equipped with Gene Scanning software (version 2.0) to detect the methylation status of four JAK-STAT pathway-related genes. Polymerase chain reaction (PCR) comprised a mixture of 0.6 μL of bisul tetreated DNA (12 ng), 0.25 μL of each forward and reverse primer (10 mM), 5 μL of LightCycler 480 High Resolution Melting Master Mix (Roche), 1.4 μL MgCl 2 (25 mM) and 2.5 μL of polymerase chain reaction PCR-grade water in a 10 µL volume in a 96-well plate. Commercially available human whole genomic methylated and unmethylated DNA samples (Zymo Research) were prepared as a set of standards (100%, 5%, 2%, 1%, and 0%) and used to semi-quantitatively measure methylation levels in the samples.
Additionally, DNA-free water was used as a blank control in each plate and all reactions were performed in duplicate for quality control. Two investigators independently assessed the melting curves, and a third investigator joined the discussion if disagreements existed.
Additionally, DNA-free water was used as a blank control in each plate and all reactions were performed in duplicate for quality control. Two investigators independently assessed the melting curves, and a third investigator joined the discussion if disagreements existed.