Epityper software v1

Quantitative methylation analysis was performed by the Sequenom's MassARRAY system (Sequenom Inc, San Diego, California, USA), which employs matrix-assisted laser desorption/ionization-time of-flight mass spectrometer (MALDI-TOF-MS). Bisulfite-treated DNA was then subjected to the following reactions: PCR amplification (94°C for 4 min; 45 cycles of 94°C for 20s, 56°C for 30 s, and 72°C for 1 min; then 72°C for 3 min); dephosphorylated by shrimp alkaline phosphatase (SAP) (Sequenom) (37°C for 20min, 85°C for 5min); followed by base specific cleavage (37°C for 3h). The resultant products were purified by the clean resin (Sequenom) and spotted on a 384-element silicon chip (SpectroCHIP, Sequenom, USA). Mass spectra were obtained using MassARRAY mass spectrometer and analyzed using EpiTYPER software v1.2 (Sequenom Inc, San Diego, California, USA) and the results were shown as percentages of methylation of each CpG site. The results were normalized by the methylation level of the test standard within the methylation analysis kit (Sequenom, Inc., San Diego, USA). Each sample was subjected to duplicate independent analyses and poor-quality and non-applicable data were eliminated in the calculations.