Cation exchange resin

The genomic DNA was isolated from blood clot using TIANamp Blood Clot DNA Kit (Tiangen biotech Co. Ltd, Beijing, China) according to manufacturer's protocol and stored at –80°C for genotyping. All samples were genotyped using matrix-assisted laser desorption ionization-time of-flight mass spectrometry (Sequenom Inc., San Diego, CA). Amplification primers and extension primers were designed using theAssayDesigner3.1 software (Sequenom Inc). Polymerase chain reaction (PCR) amplification of target sequence was performed in a multiplex reaction containing 1 μL genomic DNA. After PCR amplification, remaining dNTPs were dephosphorylated by shrimp alkaline phosphatase (Sequenom Inc.). Then, extension primers were used for locus-specific single-base extensions. The extension products were purified by cation-exchange resin (Sequenom Inc.), transferred onto the 384-well spectroCHIP (Sequenom Inc.) and genotyped using a matrix-assisted laser desorption ionization-time of-flight mass spectrometer. Genotyping data were analyzed using the MassARRAYTyper software version 3.4 (Sequenom Inc.). The full research processes are shown in Figure 1.

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).