Pyrosequencing
A pyrosequencing assay was also used for the measurement of PTPRTpromoter methylation. For that purpose, the bisulfite conversion of 1μg
of DNA using the EpiTect Bisulfite Kit (Qiagen) was followed by a PCR
targeting a fraction of PTPRT promoter. Finally, the
pyrosequencing took place in a PyroMark Q96 MD sequencer (Biotage,
Qiagen), taking advantage of the PyroMark Q96 reagents and buffers and
following the manufacturer’s instructions. Tumor or healthy tissue DNA
from PTPRT mutation carriers was used for this assay, while
commercial high-methylated and non-methylated DNA was used as a control
(CpGenome Human Methylated/Non-Methylated DNA Standard, Millipore). The
PCRs and subsequent pyrosequencing were performed in triplicates, and
the relative methylation (%) and standard deviation were represented.
The sequences of the PCR and biosynthesis primers are shown in
Supplementary Table S2.