From a review of the current literature, we selected four candidate genes to examine the 5-LOX pathway (ALOX-5, FLAP, LTA4-H and LTC4-S). Next, 22 SNP markers were selected based on previous reported associations with cardiovascular disease, with preference for coronary artery disease, and a confirmed minor allele frequency (MAF) > 0.05 in Caucasians.
From whole blood, DNA was extracted using Kleargene™ XL DNA extraction kit (LGC Genomics, Queens Road, Teddington, Middlesex, UK). Next, contaminants were removed by washing and DNA was subsequently eluted into a low salt buffer. Extracted DNA were stored at -20°C.
SNP genotyping was performed by LGC Genomics using the commercially available KASP™ genotyping assay. KASP is based on a competitive, allele specific PCR genotyping technique with a homogenous fluorescent based reporting system [37 (link)]. The reaction mix was aliquoted to standard 96-well plates containing DNA-samples from the study cohort, including at least one "no template control" per plate. PCR was performed and the fluorescent signal was analysed using a BMG PHERAstar plate reader (BMG Labtech Ltd., Aylesbury, UK). The analysis was performed according to the protocol provided by LGC Genomics [38 ]. SNP alleles correspond to the positive/forward DNA-strand according to dbSNP, human assembly GRCh38.p2 [39 ].
From whole blood, DNA was extracted using Kleargene™ XL DNA extraction kit (LGC Genomics, Queens Road, Teddington, Middlesex, UK). Next, contaminants were removed by washing and DNA was subsequently eluted into a low salt buffer. Extracted DNA were stored at -20°C.
SNP genotyping was performed by LGC Genomics using the commercially available KASP™ genotyping assay. KASP is based on a competitive, allele specific PCR genotyping technique with a homogenous fluorescent based reporting system [37 (link)]. The reaction mix was aliquoted to standard 96-well plates containing DNA-samples from the study cohort, including at least one "no template control" per plate. PCR was performed and the fluorescent signal was analysed using a BMG PHERAstar plate reader (BMG Labtech Ltd., Aylesbury, UK). The analysis was performed according to the protocol provided by LGC Genomics [38 ]. SNP alleles correspond to the positive/forward DNA-strand according to dbSNP, human assembly GRCh38.p2 [39 ].