Taqman fluorogenic 5 nuclease assay

Genomic DNA was prepared from blood samples with the QuickGene-mini80 (FUJIFILM, Tokyo, Japan). In a subset of controls (n = 160), DNA was extracted from saliva using the Oragene DNA collection kit (DNA Genotek, Kanata, Ontario, Canada). Genotyping of rs3782025 was performed with the ABI PRISM SNaPShot Multiplex kit (ABI, Foster City, CA, USA) according to the manufacturer’s recommendations. Analyses were conducted using the GeneMapper software (version 4.0; Applied Biosystems, USA). Genotyping of the remaining nine SNPs (rs1062613, rs1176713, rs3758987, rs1176744, rs6766410, rs6807362, rs6443930, rs1000952, and rs7627615) was performed with the TaqMan fluorogenic 5′ nuclease assay (ABI, Foster City, CA, USA) according to the manufacturer’s instructions. Primer sequences and assay IDs are shown in Supplementary Table S2.

We used the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA) to extract genomic DNA from samples of the participants’ peripheral blood lymphocyte. We analyzed 18 genes (AhR, ANKK1, CAT, COMT, CYP1A1, CYP1B1, CYP2B6, EPHX1, GSTP1, HSPA1L, MPO, MTHFR, NAT2, NOS3, NQO1, PON1, PTGS2, and SOD2) that are related to oxidative stress, and genotyped 47 SNPs from these genes. The SNPs were selected based on a priori knowledge of being associated with oxidative stress status, a previous study from the National Center for Biotechnology Information, as well as minor allele frequencies of ≥5 % for the Japanese and Chines populations in the HapMap (http://hapmap.ncbi.nlm.nih.gov/) to consider public health implications. Polymorphisms in ANKK1, CAT, CYP1B1, EPHX1, HSPA1L, MPO, NOS3, PON1, PTGS2, and SOD2 were identified using a TaqMan fluorogenic 5’ nuclease assay (ABI, Foster City, CA, USA); polymorphisms in AhR, COMT, CYP1A1, CYP2B6, MTHFR, NAT2, and NQO1 were identified using the Sequenom Mass ARRAY platform [25 ]; and polymorphisms in GSTP1 were identified using a multiplex polymerase chain reaction method [26 (link)]. Among the 47 genotyped SNPs, we excluded one SNP (rs2965753) from the present analyses because it was not within the Hardy-Weinberg equilibrium (P = 9.3126 × 10^-7 using the chi square test).

The genotyping of SNP which met Bonferroni correction for genome-wide significance of severe CAC in the discovery set was screened using the TaqMan fluorogenic 5’ nuclease assay (ABI, Foster City, CA, USA) in the replication set. The final volume of polymerase chain reaction (PCR) was 5 ul, containing 10 ng of genomic DNA and 2.5 ul of TaqMan Universal PCR Master Mix, with 0.13 ul of 20X Assay Mix (Assay ID C___2684958_10). Thermal cycle conditions were as follows: 50°C for 2 min to activate the uracil N-glycosylase and to prevent carry-over contamination, 95xC for 10 min to activate the DNA polymerase, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. All PCR were performed using 384-well plates by a Dual 384-Well GeneAmp PCR System 9700 (ABI, Foster City, CA, USA), and the endpoint fluorescent readings were performed on an ABI PRISM 7900 HT Sequence Detection System (ABI, Foster City, CA, USA). Duplicate samples and negative controls were included to ensure accuracy of genotyping.

The genotypes of rs3825017 in SLC22A12 and rs16890979 in SLC2A9 were screened using the TaqMan fluorogenic 5′ nuclease assay (ABI, Foster City, CA, USA). The final volume of the polymerase chain reaction (PCR) was 5 μL, and each reaction contained 10 ng genomic DNA, 2.5 μL TaqMan Universal PCR Master Mix, and 0.13 μL 20× Assay Mix. The thermal cycle conditions were as follows: 50 °C for 2 min to activate the uracil N-glycosylase and to prevent carry-over contamination, 95 °C for 10 min to activate the DNA polymerase, followed by 45 cycles at 95 °C for 15 s and 60 °C for 1 min. All of the PCR reactions were performed using 384-well plates on a Dual 384-Well GeneAmp PCR System 9700 (ABI). The endpoint fluorescent readings were performed on an ABI PRISM 7900 HT Sequence Detection System (ABI). Duplicate samples and negative controls were included to ensure the accuracy of genotyping, which was performed at DNA Link Inc. in Seoul, Korea. The distribution of the two SNPs in the study subjects satisfied Hardy–Weinberg equilibrium.

We performed GWAS in the discovery set (n = 400) with a case–control study between the subjects with severe CAC (>90th percentile for age and sex) and control (≤50th percentile for age and sex). The results were verified using the validation test. The SNP determined to have genome-wide significance after Bonferroni correction in the discovery set was screened using the TaqMan fluorogenic 5’ nuclease assay (ABI, Foster City, CA, USA) in the replication set (n = 1288). A p-value less than 0.05 was considered significant in the replication set.
Logistical regression analyses were used to calculate the odds ratios (OR), 95% confidence intervals (CI), and corresponding p-values of each SNP of severe coronary calcification, controlling for age, sex, hypertension, and diabetes as covariates with additive models. All analyses were two-tailed, and p-values <0.05 were considered statistically significant. Statistical tests were performed using PLINK version 1.9 (https://www.cog-genomics.org/plink2; Free Software Foundation Inc., Boston, MA, USA). R statistical software package, version 3.1.1 (R development Core Team; R Foundation for Statistical Computing, Vienna, Austria) was used to analyze statistical data and to draw the Manhattan plot of -log10. In the regional plot, hg19/1000 Genomes Nov 2014 ASN was used as the reference panel for linkage disequilibrium [19 (link)].