Spectrochip bioarray

Genomic DNA was extracted from the peripheral blood of each patient with the TIANGEN Kit (DP304, Beijing TIANGEN Biotechnology Co., Ltd.). The SNP genotyping method for all samples was based on the Sequenom MassARRAY® platform, and all amplification primers of the 6 NSE gene SNPs were designed according to the dbSNP database for sequencing and amplified by multiplex PCR to obtain site-specific PCR. The initial multiplex PCR amplification was performed using ABI Veriti-384 PCR. SAP (shrimp alkaline phosphatase) was used to remove the free dNTPs from the reaction system. After SAP treatment, the extended mixture was added in the abovementioned PCR system, to carry out single-base extension reactions, and then, a resin purification was conducted. Finally, a MassARRAY Nanodispenser (Agena, Inc.) was used to transfer the PCR products to the 384-well SpectroCHIP® bioarray. The genotypes and alleles were detected by the mass spectrometer MassARRAY Analyzer 4.0 and analyzed by the mass spectrometer MassARRAY TYPER4.0. According to the results of the analyses, the 6 SNPs were separately genotyped in all the samples.

Instructions to extract cells or tissues using the DNA extraction kit (BioTeKe Corporation) were observed. The DNA samples to be tested were processed using a commercial NaHSO₃ kit (ZYMO). The gene fragments to be detected were enriched and amplified by PCR reaction, and the product length was 200 to 700 bp. The PCR product was treated with shrimp alkaline phosphatase to remove free deoxyribonucleoside-5′-triphosphates (dNTPs) in the system. A transcriptase digestion reaction was followed by resin purification. The purified products of the resin were transported to a 384-well SpectroCHIP® bioarray (Agena, Inc.) using an Agena NanodispenserRS1000 dot sampling instrument (Agena, Inc.). EpiTYPER™ software provided advanced and convenient quantitative analysis of DNA methylation.