Massarray nanodispenser

10 nL of the resultant cleavage reactions was spotted onto silicon matrix‐preloaded chips (Spectro‐CHIP; Sequenom) using a MassARRAY nanodispenser (Sequenom) and analyzed using the MassARRAY Compact System matrix‐assisted laser desorption/ionization‐time‐of‐flight mass spectrometer (MALDI‐TOF) (Sequenom). The spectra's methylation ratios were calculated using epityper software v1.0 (Sequenom). The method yields quantitative results for each of the sequence‐defined analytic units referred as CpG units, which may contain either one individual CpG site or an aggregate of downstream CpG sites. Triplicate independent analyses from sodium bisulfite‐treated DNA sample were undertaken.
The effectiveness of the entire experimental procedure was assayed by analyzing as control CpGenome Universal Unmethylated DNA (Chemicon) and CpGenome Universal Methylated DNA (Chemicon, Millipore, Germany) in serial mixtures of methylated and unmethylated products, with 10% methylation increments.
Data quality control and filtering were carried out by the removal of the CpG dinucleotides whose the measurement success rate was <80%. Poor‐quality and nonvaluable data for the quantitative methylation of each CpG unit measured by MALDI‐TOF‐MS were excluded.

Peripheral venous blood (2 ml) was collected from each participant, and then genomic DNA was isolated using a WizardVR genomic DNA purification kit (Promega, Madison, U.S.A.) following the supplier’s manual. The concentration and purity of the genomic DNA were estimated on NanoDrop using two optical density wavelengths 260 and 280 nm.
Genotyping was done by MALDI-TOF-MS using a MassARRAY system (Sequenom, San Diego, CA, U.S.A.) as previously described [13 (link)]. Complete genotyping reactions were spotted on to a 384-well spectroCHIP (Sequenom) using MassARRAY nanodispenser (Sequenom) and analyzed by MALDI-TOF-MS. Genotype callings were done in real time with MassARRAY RT 3.1 and analyzed on MassARRAY Typer 4.0 (both Sequenom). For quality control, 10% of the randomly selected samples were analyzed repeatedly.

Venous blood samples (2 ml each) were collected in tubes containing EDTA for genotyping analysis. Genomic DNA was extracted from peripheral blood using a QIAamp DNA blood mini-kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The concentration and purity of the extracted DNA were detected using NanoDrop at two optical density wavelengths 260 and 280 nm.
Rs2576178 is close to 5′-UTR of Renalase gene, which may affect the binding of transcription factors. This single nucleotide polymorphism (SNP) would cause the change from T to C nucleotides and has a minor allele frequency of 0.3442. Genotyping was done by matrix-assisted laser-desorption ionization (MALDI)/time of flight (TOF)-mass spectrometry (MS) using a MassARRAY system (Sequenom, San Diego, CA, U.S.A.). Complete genotyping reactions were spotted on a 384-well spectroCHIP (Sequenom) using a MassARRAY nanodispenser (Sequenom) and analyzed by MALDI-TOF-MS. Genotype calling was done in real time with MassARRAY RT 3.1 and analyzed using MassARRAY Typer 4.0 (both Sequenom). For quality control, repeated analyses were undertaken on 10% of randomly selected samples, and three positive and three negative controls were used. Accuracy of genotyping was determined by evaluating the genotype concordance between duplicate samples.

Venous blood (2 mL each) was sampled in tubes containing ethylenediamine-tetraacetic acid (EDTA) and stored at –80°C before use. Genomic DNA was extracted using a QIAamp DNA blood mini kit (Qiagen, Germany), and the concentration and purity were estimated using NanoDrop (Thermo Electron Corp., USA) at two absorbance wavelengths of 260 and 280 nm. Genotyping was done by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) using a MassARRAY system (Sequenom, USA). Completed genotyping reactions were spotted onto a 384-well spectroCHIP (Sequenom) using a MassARRAY nanodispenser (Sequenom) and analyzed by MALDI-TOFMS. Genotype calling was done in real time with MassARRAY RT 3.1 (Sequenom) and analyzed on MassARRAYTyper 4.0 (Sequenom). For quality control, 10% of randomly-selected samples were analyzed repeatedly.

Blood samples were collected using vacutainers and transferred to test tubes containing ethylenediamine tetra-acetic acid (EDTA). Genomic DNA was isolated from the lymphocytes of whole blood samples using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). For quality control, all sample DNAs were conducted polymerase chain reaction (PCR), analyzed on a 3% agarose gel and visualized by ethidium bromide staining. IL-1A rs1800587 C/T, IL-1B rs16944 G/A, IL-6 rs1800796 C/G, IL-10 rs1800872 A/C and IL-10 rs1800896 A/G genotyping was done by MALDI-TOF MS support from CapitalBio Corporation (Beijing, China), using the MassARRAY system (Sequenom, San Diego, CA, USA) as previously described [22 (link)]. LMCAD and MPCAD at a proportion of ≈1:2 were assayed. Completed genotyping reactions were spotted onto a 384-well spectroCHIP (Sequenom) using a MassARRAY Nanodispenser (Sequenom), and analyzed by MALDI-TOF-MS (Sequenom, San Diego, CA, USA). Genotype calling was done in real time with MassARRAY RT software (version 3.1; Sequenom), and analyzed using MassARRAY Typer software (version 4.0; Sequenom). For IL-6R rs7529229 T/C and IL-8 rs4073 T/A, genotyping study was performed using the LDR method [23 (link),24 (link)], with technical support from Shanghai Biowing Applied Biotechnology Company (Shanghai, China).

We selected SNPs (rs8051542, rs12443621, rs3803662, rs4784227 and rs3112612) in the TOX3/LOC643714 locus at 16q12.1 from the confirmatory results from GWAS or meta-analysis in multiple populations [6 (link),7 (link),11 (link),20 (link)].
The five SNPs were genotyped using the SEQUENOM MassARRAY matrix-assisted laser desorption ionization-time of flight mass spectrometry platform (Sequenom, San Diego, CA, USA). Primers for multiplex PCR and extended reactions were designed using proprietary software (Assay Designer, version 3.1) provided by Sequenom Inc (San Diego, CA, USA). In accordance with the manufacturer’s instructions, SNPs were genotyped using Sequenom MassARRAY genotyping technology (Sequenom, San Diego, CA, USA) and amplified in multiplex PCR by a standard PCR protocol. The genomic amplification product was cleaned using shrimp alkaline phosphatase (Sequenom, San Diego, CA, USA) to neutralize any unincorporated dNTPs, followed by a single-base extension reaction using the iPLEX enzyme (Sequenom, San Diego, CA, USA) and mass-modified terminators (Sequenom, San Diego, CA, USA). The products of the iPLEX reaction were desalted and transferred onto a SpectroCHIP (Sequenom, San Diego, CA, USA) by the MassARRAY nanodispenser (Sequenom, San Diego, CA, USA), which was then analyzed by the MassARRAY analyzer by combining base calling with the clustering algorithm.