The previous study reported the criteria for candidate genes and SNPs related to the synthesis, transport, and catabolism of vitamin D [18 (link)]. We first selected NADSYN1/DHCR7 (rs1790349 and rs12785878), GC (rs222040, rs1155563, rs16846876, rs2298849, rs7041, and rs4588), CYP24A1 (rs2209314), CYP2R1 (rs10741657), and VDR (rs2228570, rs7975232, and rs757343) as candidate SNPs. Based on the Hardy–Weinberg equilibrium (r2 > 0.8) and the genotype success rate (>95%), five transport SNPs (GC-rs1155563, GC-rs16846876, GC-rs2298849, GC-rs7041, and GC-rs4588), and one catabolism SNP (CYP24A1-rs2209314) were selected. SNP genotyping was performed using the Sequenom MassARRAY iPLEX Gold platform (Sequenom, San Diego, CA, USA).
Massarray iplex gold platform
Manufactured by Labcorp
Sourced in United States
The MassARRAY iPLEX Gold platform is a high-throughput, mass spectrometry-based genotyping system. It is designed for the analysis of genetic variations, including single nucleotide polymorphisms (SNPs) and small insertions/deletions.
Automatically generated - may contain errors
Lab products found in correlation
Rapid dna extraction and detection kit, by Tiangen Biotech (2 mentions)
Flexi gene dna kits, by Qiagen (1 mentions)
Puregene genomic dna purification kit, by Qiagen (1 mentions)
Masterpure dna purification kit for blood version 2, by Illumina (1 mentions)
Qiaamp dna blood biorobot mdx kit, by Qiagen (1 mentions)
Autopure ls machine, by Qiagen (1 mentions)
Compact maldi tof mass spectrometer, by Labcorp (1 mentions)
7 protocols using massarray iplex gold platform
1
Vitamin D Pathway Genetic Markers
2
Genetic Predictors of Vitamin D Metabolism
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Single nucleotide polymorphisms (SNPs) involved in vitamin D synthesis and metabolism pathways were selected according to previous studies reported among pregnant or non‐pregnant population. Besides, these SNPs locate in the functional domain of genes and play a vital role in vitamin metabolism included in the database of NCBI VDR (rs10735810, rs3847987, rs1544410, rs7968585, rs2408876, rs10783219); GC (rs7041, rs4588, rs2282679, rs1155563, rs12512631, rs222020, rs16847015, rs17467825, rs2070741, rs2298849, rs16846876, rs842999, rs222035, rs3755967, rs11939173, rs2298850, rs12512631); CYP2R1 (rs2060793, rs10741657, rs1993116, rs7116978, rs12794714, rs1562902, rs10766197, rs10500804, rs10766197, rs10877012, rs11023374); CYP24A1 (rs6013897, rs927650, rs2248137, rs6068816, rs73913757, rs2209314, rs2762939, rs17470271); CYP27B1 (rs4646536, rs703842, rs10877012, rs118204009); CYP3A43 (rs680055, rs2242480); NADSYN1/DHCR7 (rs3829251, rs1790349, rs12785878, rs7944926, rs12800438, rs3794060, rs4945008, rs4944957); and RXRA (rs9409929, rs11185644) were selected as candidate SNPs. DNA was extracted from the peripheral blood leukocytes using a Rapid DNA Extraction and Detection Kit (Tiangen) according to the manufacturer's protocol, and SNP genotyping was conducted and analyzed by using the MassARRAY iPLEX Gold platform (Sequenom).
3
Vitamin D Genetics and Metabolism
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According to previous studies, relative SNPs have been selected in genes involved in vitamin D synthesis and metabolic pathways. In addition, these SNPs are located in the functional domain and play a vital role in vitamin metabolism from the NCBI database. The following were selected as candidate SNPs: VDR (rs10735810, rs3847987, rs1544410, rs7968585, rs2408876, rs10783219); GC (rs7041, rs4588, rs2282679, rs1155563, rs12512631, rs222020, rs16847015, rs17467825, rs2070741, rs2298849, rs16846876, rs842999, rs222035, rs3755967, rs11939173, rs2298850, rs12512631); CYP2R1 (rs2060793, rs10741657, rs1993116, rs7116978, rs12794714, rs1562902, rs10766197, rs10500804, rs10766197, rs10877012, rs11023374); CYP24A1 (rs6013897, rs927650, rs2248137, rs6068816, rs73913757, rs2209314, rs2762939, rs17470271); CYP27B1 (rs4646536, rs703842, rs10877012, rs118204009); CYP3A43 (rs680055, rs2242480); NADSYN1/DHCR7 (rs3829251, rs1790349, rs12785878, rs7944926, rs12800438, rs3794060, rs4945008, rs4944957); RXRA (rs9409929, rs11185644). A Rapid DNA Extraction and Detection Kit (Tiangen) was used to extract DNA from peripheral blood leukocytes according to the manufacturer's protocol, and SNP genotyping was conducted and analyzed using the MassARRAY iPLEX Gold platform (Sequenom).
4
Genotyping Protocol for Genetic Variants
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For each participant, 5 ml peripheral venous blood was collected after signing informed consent and before drug used. Genomic DNA was extracted using the phenol-chloroform method within 1–2 weeks and saved in −20°C refrigerator. SNP genotyping was performed with the Sequenom MassARRAY iPLEX Gold platform (Sequenom, San Diego, CA, United States) (Ikeda et al., 2008 (link)). Primers were designed using the MassARRAY Assay Design 3.1 software. Two amplification primer systems were built with the balance of capturing as many SNP loci as possible while limiting costs. Genomic fragments containing SNPs were amplified by polymerase chain reaction (PCR) in a total reaction volume of 5 μl, which included 20–50 ng of genomic DNA, in two 384-well plates using the ABI GeneAmp® 9700 384 Dual. Purified and specific genotyping primers were used to amplify target sites. Genotypes were automatically called by MassARRAY Typer 4.0 and verified manually. To ensure the accuracy of genotyping, 32 samples were duplicated for quality control, and no genotyping errors were found. Additionally, each 384-well plate had four blank controls. Any individual whose missing genotypes were greater than 50% was excluded from further statistical analysis.
5
Genome-wide Genotyping and Validation of Central China Cohort
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The genotyping in the discovery stage for the central China cohort was conducted by Illumina 610-Quad Human Beadchip array (Illumina, Inc., San Diego, CA, USA). The genomic DNA was isolated from peripheral blood mononuclear cells (PBMCs) with standard procedures using Flexi Gene DNA kits (QIAGEN GmbH, Hilden, Germany) and was diluted to working concentrations of 50 ng/μl for genome-wide genotyping and 15–20 ng/μl for the validation study. The SNPs in the X chromosome for the validation stage were genotyped using the Sequenom MassArray iPlex Gold platform (Sequenom, Inc., San Diego, CA, USA).
6
DNA Extraction and SNP Genotyping
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DNA was either isolated on the BioRobot Universal with the QIAamp DNA Blood BioRobot MDx Kit (Qiagen, Hilden, Germany) and eluted in Qiagen buffer AE (10 mM Tris-Cl 0.5 mM EDTA; pH 9.0), or with the Gentra Autopure LS machine using the Puregene Genomic DNA purification Kit (Gentra Systems, Minneapolis, MN 55441 USA), or manually using the MasterPure TM DNA Purification Kit for Blood Version II (Epicentre® Biotechnologies, Madison, WI, USA). SNPs were genotyped with the iPLEX Gold massarray platform (Sequenom) at the Centre for Integrative Genetics, Norwegian University of Life Sciences, Ås, Norway.
7
Mitochondrial Haplogroup Genotyping Protocol
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