Iplex gold

We genotyped six SNPs in or near the CACNA1C gene which were chosen based on their previously reported potentials to affect gene expression/regulation as mentioned earlier. These SNPs were rs1006737, rs1051375, rs10466907, rs11062319, rs723672, and rs58619945. The LD matrix between these SNPs and their positions on chromosome are shown Fig. 2.Figure 2

Linkage disequilibrium matrix of the studied CACNA1C SNPs rs1006737, rs1051375, rs723672, rs10466907, rs11062319, and rs58619945 in Han Chinese in Beijing, China. Note: CACNA1C: calcium voltage-gated channel subunit alpha1 C; SNPs: single nucleotide polymorphisms.

Genomic DNA was extracted from whole blood according to standard procedures. Investigation of the SNPs was performed on the commercially available Sequenom MassARRAY platform. The six SNPs were simultaneously identified by a genotyping technology called iPLEX Gold (Sequenom) followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis^{45 (link), 46 (link)}.

Tag-SNPs of IL-6 and IL-6R were selected following data release from Phase II of the International HapMap project [15 (link)]. Sample based genotypes were downloaded for all variants in genomic regions including from 5,000 bp 5-prime upstream to 5000 bp 3-prime downstream of IL-6 and IL-6R independently.
Since the study populations under investigation were from the Chinese population, downloaded genotypes were restricted to those for the Han Chinese in Beijing, China (CHB) population (http://hapmap.ncbi.nlm.nih.gov). Tag-SNPs were selected using a pairwise tagging algorithm by Haploview software (available at http://www.broadinstitute.org/haploview), with a correlation coefficient (r2) exceeding 0.8 for all downloaded SNPs with minor allele frequency (MAF) >5% [16 (link)]. Because the tag-SNP probabilities were discrete, accordingly, functional ranking of tag-SNPs with the same probability was used.
Blood samples from all participants were collected and stored at −20°C. Genomic DNA was extracted from peripheral leukocytes by using a Genomic DNA Extraction kit (QIAamp DNA Blood Mini Kit; Qiagen, Hilden, Germany). MassArray (Sequenom, USA) was used for genotyping selected tag-SNPs (Gabriel et al., 2009), and this assay was accomplished by Bio Miao Biological Technology (Beijing, China). The primers were designed using iPLEX GOLD (Sequenom, USA) [17 ].

Blood samples from all participants were collected and stored at −80°C until analysis. Genomic DNA was isolated from peripheral blood leukocytes in 2‐ml aliquots of whole‐blood samples with a Qiagen blood midi kit (Qiagen, Chatsworth, CA, USA). The purified DNA samples were then stored at 4°C in sterile plastic vials. Genotype analyses for SNPs determination were performed with the single‐base extension polymerase chain reaction Sequenom iPLEX‐Gold and the mass spectrometry‐based platform MassARRAY MALDI‐TOF (Sequenom, San Diego, CA, USA) at the Spanish Genotyping National Centre (CEGEN‐ISCIII). In brief, the analyses consisted of an initial locus specific PCR, followed by single‐base extension using mass‐modified dideoxynucleotide terminators of an oligonucleotide primer, which anneals immediately upstream of the polymorphic site of interest. Using matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry, the distinct mass of the extended primer identifies the allele (Gabriel, Ziaugra, & Tabbaa, 2009). The [CAAA]^4‐5 and rs552393576 polymorphisms in the TAFP2B gene were genotyped by direct sequencing in the same reaction. Primers and PCR conditions are available upon request.