MALDI-TOF mass spectrometry

Bisulfite converted DNA of all participants was amplified by bisulfite-specific primers. The sequence of target region is shown in Additional file 1: S1. There is no single nucleotide polymorphism (SNP) nor CpG site in the primers. Forward primer: 5′-aggaagagagTAAAATGTTGGGATTATAGTTTGGG-3′, reverse primer: 5′-cagtaatacgactcactatagggagaaggctAAAACCAAATTCCTTCTTCTACACC-3′. Upper case letters presented the sequence specific regions, and the unspecific tags were shown in lower case letters. The PCR products were analyzed by a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry for the semi-quantitative measurements of the DNA methylation intensity at the single CpG resolution (Agena Bioscience, California, U.S.). Briefly, the PCR amplified products were incubated with Shrimp Alkaline Phosphatase (SAP) and further transcribed to RNA by T7 transcriptase according to the standard protocol of Agena EpiTyper assay (Agena Bioscience, California, U.S.). The RNA was digested by RNase into small fragments and then cleaned the ions by resin. The final products were dispensed on a 384 SpectroCHIP. The DNA methylation levels were semi-quantitatively determined by comparing the intensities of methylated and non-methylated segments. The data were collected by SpectroACQUIRE v3.3.1.3 software and visualized by EpiTyper v1.3 software.