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Sequenom MassARRAY quantitative DNA methylation analysis

WY Wuping Yang
KZ Kenan Zhang
LL Lei Li
YX Yawei Xu
KM Kaifang Ma
HX Haibiao Xie
JZ Jingcheng Zhou
LC Lin Cai
YG Yanqing Gong
KG Kan Gong
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Genomic DNA was extracted from ccRCC and pair-matched adjacent normal tissues, and the bisulfite conversion reaction was performed according to the manufacturer’s instructions. The PCR mixtures were pre-heated for 4 min at 94 °C, followed by 45 cycles of 94 °C for 20 s, 56 °C for 30 s and 72 °C for 1 min, the final extension at 72 °C for 3 min. PCR products were incubated with Shrimp Alkaline Phosphatase following the manufacturer’s protocol. After in vitro transcription and RNase A digestion, small RNA fragments with CpG sites were acquired for the reverse reaction. The methylation ratios of the products were calculated using Epityper software Version 1.0 (Sequenom, San Diego, CA, USA). The Sequenom MassARRAY platform (Oebiotech, Shanghai, China) was utilized to quantitatively analyze the DNA methylation status of ZNF582-AS1 DNA. PCR primers were designed using EpiDesigner, and their sequences were listed in Additional file 1: Table S1.

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