384 well spectrochip

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).

The primers used in the present study for PCR and genotyping were shown in Table 2. The genotyping of the three selected SNPs was conducted by a MALDI-TOF MS method as described previously [34 (link)]. The SNPs genotyping was performed through a MassARRAY system (Sequenom, San Diego, California, USA). The genotyping reactions were accomplished using 384-well spectroCHIP (Sequenom, San Diego, California, USA) equipped with a MassARRAY Nanodispenser. The genotype calling was carried out via MassARRAY RT software (version 3.1, Sequenom, San Diego, California, USA) and the generated data was then processed by MassARRAY Typer software (version 4.0, Sequenom, San Diego, California, USA). For quality control, ten percent of the extracted genomic DNA was sampled randomly and genotyped twice and the subsequent genotyping data demonstrated no discrepancies. In addition, all the genomic DNA samples used for genotyping were coded randomly in order to avoid bias.

Blood samples were collected by experienced nurses using vacuum tubes containing ethylenediamine tetra-acetic acid (EDTA). Then, we isolated genomic DNA from whole blood using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). Quality control of sample DNAs was conducted by performing polymerase chain reaction (PCR) and analysis on a 3% agarose gel and visualized by ethidium bromide staining. Genotyping of the single nucleotide polymorphisms (SNPs, COX-2 rs5275, rs5277, and rs689466) was performed by MALDI-TOF MS support from CapitalBio Corporation (Beijing, China) [18 (link)–20 ]. Sample transfer was completed by MassARRAY Nanodispenser (Sequenom) to a 384-well spectroCHIP (Sequenom) and then analyzed by MALDI-TOF-MS. MassARRAY RT genotype-calling software (version 3.1; Sequenom) was used to call each genotype in real time eliminating a time-consuming process.

Whole blood (3 mL) was drawn from an arm vein into a sterile tube containing ethylenediaminetetraacetic acid (EDTA) and stored at -80°C until genotype analysis was performed. The 12 SNPs of the COX pathway genes were selected from the NCBI database (http://www.ncbi.nlm.nih.gov/SNP), according to the criteria: (1) the SNPs had been examined in previous studies [14 (link)–22 (link)]; (2) the SNPs lead to amino acid changes. Genotypes of the 12 SNPs were examined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry methods according to our previous study [24 (link)]. In brief, each SNP gene possessed a specific genotype, with two amplification primers and one extension primer. The reaction mix was desalted by adding 6 mg of cation exchange resin (Sequenom Inc., San Diego, CA), mixed, and resuspended in 25 μL of water. Once the primer extension reaction was completed, the samples were spotted onto a 384-well spectroCHIP (Sequenom Inc., San Diego, CA) using a MassARRAY Nanodispenser (Sequenom Inc., San Diego, CA) and genotyped using the MALDI-TOF mass spectrometer. Genotyping was performed in real time with MassARRAY RT software, version 3.0.0.4, and analyzed using the MassARRAY Typer software, version 3.4 (Sequenom Inc., San Diego, CA).

In brief, 2mL of peripheral blood was collected in vacuum tubes containing 5% EDTA and frozen at -80 °C until use. Genomic DNA was extracted using a DNA Purification Kit (Tiangen Biotech) according to the manufacturer’s instructions. and the concentration and purity were estimated using NanoDrop at two optical density (OD) wavelengths 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, San Diego, CA, USA). Completed genotyping reactions were spotted onto a 384-well spectroCHIP (Sequenom) using a MassARRAY nano dispenser (Sequenom) and analyzed by MALDI-TOFMS. Genotype were called in real time on MassARRAY RT 3.1 (Sequenom) and analyzed on MassARRAY Typer 4.0 (Sequenom). For quality control, 10% of randomly-selected samples were analyzed repeatedly.