Matrix assisted laser desorption ionization time of flight mass spectrometry

The RelaxGene Blood DNA System DNA isolation kit (Tiangen, Beijing, China) was used for preparing genomic DNA following the recommendations of the manufacturer. The SNP rs7294 was selected to reflect the natural haplotype block for genotyping according to our previous study [34] (link). Genotyping was performed using the MassARRAY high-throughput DNA analysis system with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Sequenom, Inc., San Diego, CA, USA). The primers were designed using MassARRAY Assay Design software (version 3.1). SNPs were genotyped using iPLEX Gold technology (Sequenom) followed by automated data analysis with the TYPE RT software version 4.0. Three samples were removed due to failed genotyping.

A panel of six SNPs of PRM1, PRM2 and TNP1 were selected for study, based on previous investigations. Genotyping analysis of the SNPs selected for fast-track validation analysis was performed using Sequenom MassARRAY technology (San Diego, CA, USA). Genomic DNA (15 ng) was used to genotype each sample. Locus-specific PCR and detection primers were designed using MassARRAY Assay Design 3.0 software at the Department of Reproduction and Genetics at the affiliated Jinling Hospital of Nanjing University. DNA samples were amplified by multiplex PCRs and the amplification products were then used for locus-specific single-base extension reactions. The resulting products were desalted and transferred to a 384-elementSpectroCHIP array (Sequenom). Allele detection was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Sequenom). Mass spectrograms were analysed by MassARRAY Typer software. SNPs detected with a call rate lower than 90% in the cases and controls, or beyond the Hardy–Weinberg equilibrium in the controls (P < 0.05), were excluded.

Genotyping for TRPV1 SNPs rs222747 was performed in all patients. The MassARRAY Assay Design 3.1 software was used to design a single 20-multiplex reaction in which the SNPs of the TRPV1 gene was included. Genotyping was performed using iPLEX Gold technology (31 (link)) and MassARRAY high-throughput DNA analysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Sequenom).

EDTA-anticoagulated whole blood (5 mL) samples were collected from all patients and stored at −80°C. Genomic DNA was extracted from peripheral blood lymphocytes using the FlexiGene DNA Purification Kit (Qiagen, Hiden, Germany) according to the manufacturer's instructions and diluted to 20 ng/μL. The rs1801133 and rs1801131 single nucleotide polymorphism (SNP) of MTHFR was genotyped using SequenomMassARRAY with the SequenomMassARRAY Assay Design 3.0 software used to design the polymerase chain reaction (PCR) and detection primers. The PCR products were subsequently used as templates for locus-specific single-base extension reactions. The resulting products were desalted and transferred to a 384-element SpectroCHIP array (Sequenom Inc.). Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (Sequenom Inc.) was used for allele detection. The mass spectrograms were analyzed using the MassARRAY Type software (Sequenom Inc.). We performed quality control of SNPs and samples at a call rate of 99.7%. The genotype distributions and allele frequencies for MTHFR gene SNP rs1801131 and rs1801133 were shown in Supplementary Table 2. The genotype frequencies of all subjects comply with Hardy-Weinberg equilibrium (p > 0.05).