Methprimer

Bisulfite treatment of DNA was done using EZ DNA Methylation-Gold Kit (Zymo Research, CA, USA) per manufacturer’s instructions. Two different MS-PCR reactions were performed in 25ul volume in (PerkinElmer; USA), each reaction contains 1 × buffer, 1 × Q-solution, 1 × betaine, and 200 uM from each of dATP, dCTP, and dTTP, together with 150 uM/50 uM dGTP/7′ deaza-dGTP, 2U of Qiagen polymerase, and 500 ng of the bisulfate-treated DNA. A total of 10 pmol of two different sets of primers, designed according to Methprimer (Thermofisher scientific, USA) software, was added in two separated reactions; one set was methylated primers designed for the PCR amplification of the methylated CpG island located upstream of the repeats of PCR cycles started with denaturation at 95 °C for 5 min, then 35 cycles at 95 °C for 1 min, 65 °C for 1 min, 75 °C for 2 min, and finally extension at 75 °C for 10 min. Meanwhile, the other set was unmethylated primer pairs for PCR amplification of fragments containing the unmethylated CGG repeats of thermal cycles proceeded with denaturation of PCR reaction at 95 °C for 5 min, followed by 35 cycles of 95 °C for 1 min, 58 °C for 1 min, and 75 °C for 2 min, and then a final extension 10 min at 75 °C. PCR fragments were analyzed by electrophoresis through 2% agarose gels stained with ethidium.

BS and qMSP protocols were made available as detailed methods at protocols.io [45 ]. Primers were designed with MethPrimer and purchased from Thermo Fisher Scientific (Waltham, MA, USA); the sequences are available in Table S2.

After bisulfite conversion, we determined DNA methylation patterns in paired tumor and normal tissues collected from five Taiwanese patients using Infinium Human Methylation 450K BeadChips (Illumina, San Diego, CA, USA) for one patient and EPIC Methylation Beadchips (Illumina) for four patients. In the DNA methylation assay, beta values were used to score the methylation level. Beta values are the ratio of the intensity of the methylated signal to the overall (sum of methylated and unmethylated) signal and thus ranged from 0 (no methylation) to 1 (full methylation). To design TSTD1 methylation–specific primers, we used MethPrimer (42 (link)) and Methyl Primer Express (v1.0, Thermo Fisher Scientific).