mythelation data of low molecular weight protein

Quantitative DNA Methylation Analysis of the Isolated Genomic DNA
Using the QIAamp DNA Mini Kit (QIAGEN, Valencia, CA), genomic DNA was purified according to the protocols. Then, methylation levels were analyzed to assess CpG content in breast tissue DNA by using Sequenom MassARRAY (San Diego, CA). According to the manufacturer's instructions, specific primers for target genes were used to evaluate the methylation at CpG sites. PCR was performed with Hot Start Taq DNA polymerase (0.2 U), primers (0.2 mM), dNTPs (200 mM), and bisulfite-treated DNA (10 ng) in a 5 µL reaction system. The amplification cycle included an initial denaturation at 94°C for 15 min, followed by 45 cycles of 94°C for 20 s, 56°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 3 min. In order to remove the unincorporated dNTPs, 2µL premix containing shrimp alkaline phosphatase (SAP) was added to the final system, followed by incubating at 37°C for 40 min, and then heat inactivation at 85°C for 5 min. The products were then used for transcription and RNase A cleavage. The final products were spotted on a 384-pad Spectro-CHIP (Sequenom) and analyzed with a mass spectrometer (MassARRAY MALDI-TOF). Using Sequenom EpiTYPER application, methylation levels were quantified.