384 spectrochip

Differential methylation of Cytosine-phosphate-guanines (CpG) was validated using MassARRAYEpiTYPER assays (Sequenom, San Diego, CA, USA). We designed three primer sets by EpiDesigner software (http://epidesigner.com) to cover MVPs of gene ALOX15, APOB, and CES1 (Supplementary Table S6). In total, 6 CpGs of ALOX15, 38 CpGs of APOB, and 13 CpGs of CES1 were included in the target products, respectively. Each reverse primer was designed to contain a T7 promoter tag for in vitro transcription, and each forward primer incorporated a 10-mer tag to adjust for melting temperature differences. According to the manufacturer’s standard protocol (Sequenom), bisulfite-converted genomic DNA was prepared for polymerase chain reaction (PCR). Amplification parameters were set as follows: 95°C for 5 min, 94°C for 20 sec, 60°C for 25 sec, and 72°C for 1 min for a total of 40 cycles, with a final incubation at 72°C for 5 min. PCR products were used in in vitro transcription reactions (T-cleavage assay). Samples were then spotted on a 384-SpectroCHIP (Sequenom) followed by spectral acquisition on a MassARRAY analyzer compact MALDI-TOF-MS (Sequenom). Methylation data of individual units (one to three CpG sites per unit) was generated by the Epitope software (Sequenom). The non-applicable reading and its corresponding site were eliminated in calculations.

For methylation analysis, the Sequenom MassARRAY EpiTYPER Assay was applied as described previously [61 (link)]. The PCR amplicons were conducted subsequently according to the protocol of the Sequenom EpiTYPER Assay and cleaned by Resin. A nanodispenser was used to transfer the products to a 384 SpectroCHIP (SEQUENOM, San Diego, CA, USA). The chips were read by a Sequenom Mass Spectrometer system (SEQUENOM, San Diego, CA, USA). Data was gathered by SpectroACQUIRE v3.3.1.3 software (SEQUENOM, San Diego, CA, USA) and visualized with MassArray EpiTYPER v1.0 software (SEQUENOM, San Diego, CA, USA). Results were depicted as “beta” values (β) between 0 and 1.