Peripheral venous blood was obtained from each subject, and genomic DNA was extracted using a DNA extraction kit (Simgen, Hangzhou, China) according to the manufacturer’s protocols. Genomic DNA concentration was determined using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). The rs7356506 single nucleotide polymorphism (SNP) in the CFI gene was genotyped in our study. The SNP was genotyped using the Sequenom MassARRAY technology platform with iPLXGOLD chemistry (Sequenom, San Diego, CA). Genotyping reagents recommended by the iPLEX Gold SNP genotyping kit (Sequenom) were used in this study. The software and equipment provided by the MassARRAY platform (Sequenom) were also employed.
Massarray technology platform
Manufactured by Labcorp
Sourced in United States
The MassARRAY technology platform is a high-throughput, mass spectrometry-based system that enables the analysis of genetic variations and molecular signatures. It is designed for efficient and accurate genotyping, gene expression analysis, and other applications in the field of molecular biology and genetics.
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Lab products found in correlation
Axyprep blood genomic dna miniprep kit, by Corning (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Massarray platform, by Labcorp (1 mentions)
Sequence detection software, by Thermo Fisher Scientific (1 mentions)
Abi prism 7500, by Thermo Fisher Scientific (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
Iplex gold chemistry, by Labcorp (1 mentions)
Iplex gold reagent set, by Labcorp (1 mentions)
Typer software version 4.0, by Labcorp (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (1 mentions)
Massarray nanodispenser, by Labcorp (1 mentions)
Iplex gold reagent kit, by Labcorp (1 mentions)
Massarray assay design 3, by Labcorp (1 mentions)
Spectrochip array, by Labcorp (1 mentions)
Hotstartaq dna polymerase, by Qiagen (1 mentions)
Tianamp blood clot dna kit, by Tiangen Biotech (1 mentions)
11 protocols using massarray technology platform
1
CFI Gene Polymorphism Genotyping
2
Genotyping of Immune-Related Variants
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According to the results of GWAS, six highly significant variants (P <1 × 10−5) were selected for further validation. To identify more potential variants, we also focus on SNPs (P <1 × 10−2) in coding region. Considering the function of genes, 23 exonic SNPs of immune-related genes were selected. Therefore, a total of 29 variants were included in the subsequent verification analysis. In addition, we also validate nine previously reported SNPs associated with the responsiveness to influenza vaccination. And the minor allele frequencies (MAFs) of these nine SNPs were no less than 0.05 in the Chinese Han population according to the 1000 Genomes Project data. Genotyping of each SNP was conducted using the MassARRAY technology platform (Sequenom, San Diego, California, USA) and determined by BioMiao Biological Technology (Beijing, China). Genotyping was performed by technicians who were blinded to the study design. According to the number of mutations carried by the subjects, their genotypes were classified into wild-type homozygote, mutant heterozygote, and mutant homozygote. SNPs with HWE-P <0.001 or call rate <95% were excluded.
3
Robust Genotyping Techniques for Variant Analysis
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Genotyping was performed by the Sequenom MassARRAY technology platform using iPLEX GOLD chemistry (Sequenom, San Diego, CA). Briefly, specific assays were designed using the MassARRAY AssayDesign software package with filtering of proximal SNPs and checking of specificity for PCR amplification and the subsequent primer extension reaction. SpectroTyper 4.0 was used to call genotypes automatically, followed by manual review.
As a secondary platform, PCR-based TaqMan assays (Applied Biosystems, Foster City, CA) were performed according to the manufacturer’s instructions, and the results were analyzed on the ABI Prism 7500 using Sequence Detection Software (Applied Biosystems Co. Ltd., USA). To confirm the accuracy of the genotyping results, 10% of the samples of each SNP were randomly selected to be tested twice by different lab personnel; the reproducibility was 100%.
As a secondary platform, PCR-based TaqMan assays (Applied Biosystems, Foster City, CA) were performed according to the manufacturer’s instructions, and the results were analyzed on the ABI Prism 7500 using Sequence Detection Software (Applied Biosystems Co. Ltd., USA). To confirm the accuracy of the genotyping results, 10% of the samples of each SNP were randomly selected to be tested twice by different lab personnel; the reproducibility was 100%.
4
Genetic Factors in Severe COVID-19
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We summarized the genomic loci related to severe COVID-19 reported in the previous GWASs or related to the severity of other viral infectious diseases, and twenty-two SNPs in IFN signaling genes and FOXP4 were selected. The minimum allele frequencies (MAFs) of all SNPs were greater than 0.05 in the Chinese population according to the SNP database in NCBI (https://www.ncbi.nlm.nih.gov/snp/ ). These SNPs of IFN signaling genes including: TLR3 rs3775291 and rs5743313, TLR7 rs3853839, DDX58 rs3739674 and rs10813831, IFIH1 rs1990760 and rs2111485, IFNAR2 rs2236757, rs13050728, rs1051393 and rs2229209, TYK2 2304256, MX1 rs17000900 and rs2071430, OAS1 rs10774671, rs1131454, and rs2660, OAS3 rs10735079, rs2285933 and rs1859330. Moreover, rs1886814 and 2894439 in the FOXP4 locus were selected, significantly associated with severe COVID-19 in European populations, and meta-analyses with multiple populations. Basic information regarding the SNPs was demonstrated in Supplementary Table 1 .
Genotyping of each SNP was performed using the MassARRAY technology platform (Sequenom, San Diego, California, USA) and determined by BioMiao Biological Technology (Beijing, China).
Genotyping of each SNP was performed using the MassARRAY technology platform (Sequenom, San Diego, California, USA) and determined by BioMiao Biological Technology (Beijing, China).
5
Genotyping SENP Polymorphisms in Whole Blood
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Peripheral blood samples were collected from each subject into a test tube containing EDTA as anticoagulant. Genomic DNA was isolated from the whole blood using the BioTECH Blood Genomic DNA Miniprep Kit (Beijing, China) according to the manufacturer's directions. The polymorphisms of SENP1 rs61918808, SENP2 rs6762208, and SENP7 rs61697963 were genotyped using the Sequenom MassARRAY technology platform with the complete iPLEX Gold Reagent Set (Sequenom, CA) in the conditions recommended by the manufacturer. Assay data were analyzed using Sequenom TYPER software (version 4.0). The primers were designed by ADS software 2.0 (Agena Bioscience, CA). The primer sequences used for genotyping were listed in Table 1 .
6
Genetic Variants in Obesity Genes
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Eleven tag SNPs in LEP, LEPR, and PPARG were selected using dbSNP database (https://www.ncbi.nlm.nih.gov/snp/ ) and SNPinfo Web Server (https://manticore.niehs.nih.gov/ ) with minor allele frequency (MAF) > 0.05 in the Chinese Han Beijing (CHB) population and r2 > 0.8, including rs2167270 and rs11761556 in LEP, rs1327118, rs7602, rs1137101, rs1938489, rs6673591, rs1137100, and rs13306523 in LEPR, and rs796313 and rs17793951 in PPARG (Supplementary Table 1 ). Genome DNA was extracted using an AxyPrep Blood Genomic DNA Miniprep kit (Axygen, Union City, California, United States) according to the manufacturer’s instructions. Genotyping of candidate SNPs was conducted using the MassARRAY technology platform (Sequenom, San Diego, California, United States) and implemented by BioMiao Biological Technology (Beijing, China). As shown in Supplementary Table 2 , the call rates for 11 tag SNPs in LEP, LEPR, and PPARG were all higher than 98.0%. Additionally, we used HaploReg (V.4.1, https://pubs.broadinstitute.org/mammals/haploreg/haploreg.php ) to explore protein binding annotations of SNPs.
7
Genotyping of GAB2 rs2373115 Polymorphism
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5 ml peripheral venous blood samples of all subjects were collected in a heparin vacuum collector before treatment, and the genomic DNA in peripheral blood was extracted using a DNA extraction kit. SNP genotyping was performed using the MassARRAY technology platform (Sequenom, CA, USA) and determined by Bomiao Biotechnology Co., Ltd. (Beijing, China). The primer sequences for GAB2 rs2373115 were as follows: forward: 5'-CGACAGAGCGAGACTTCG'-3, and reverse: 5'-GTAGGCAAATATGGACAA-3'. The polymerase chain reaction (PCR) included 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and the last cycle for 10 min. The PCR products were horizontally evaluated by agarose gel electrophoresis (2% w/v) and ethidium bromide staining. More than 1/10 of the samples were used for direct sequencing to test the repeatability, and the repeatability was 100% in our study [26 (link)].
8
Genotyping of IL-10 and IL-10RA SNPs
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Seven tag SNPs (four SNPs including rs1800871, rs1800896, rs3021094 and rs3790622 in IL-10; three SNPs including rs2282494, rs2508450 and rs4252249 in IL-10RA) were selected using GVS: http://gvs.gs.washington.edu/GVS147/and SNPinfo: http://snpinfo.niehs.nih.gov/ with minor allele frequencies (MAFs) >0.05 in CHB and r2>0.8. Genomic DNA was extracted using an AxyPrep Blood Genomic DNA Miniprep kit (Axygen, Union City, California, USA) according to the manufacturer’s instructions. Genotyping of each SNP was conducted using the MassARRAY technology platform (Sequenom, San Diego, California, USA) and determined by BioMiao Biological Technology (Beijing, China). The call rates for SNPs in IL-10 rs1800871, rs1800896, rs3021094 and rs3790622, and IL-10RA rs2282494, rs2508450 and rs4252249 were 99.6%, 99.4%, 99.1%, 99.6%, 95.1%, 99.5% and 99.6%, respectively.
9
Profiling Somatic Mutations in FFPE Samples
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The genomic DNA was extracted from 6-μm-thick slides per FFPE block and purification of genomic DNA was performed using a QIAamp DNA FFPE tissue kit (#56404, Qiagen, Hilden, Germany). Each genomic DNA sample was eluted in 50 μL of DNase- and RNase-free water, quantified using the Quant-iT PicoGreen dsDNA Assay kit (Invitrogen/Life Technologies, Grand Island, NY), and normalized to a 5 ng/μL concentration. High-throughput profiling of somatic mutations that span 471 unique mutation sites in 41 oncogenes and tumor suppressor genes was performed using OncoMap version 4.4 Core (OncoMap_v4.4C) under the Sequenom MassARRAY technology platform (Sequenom, San Diego, CA) [4 (link),5 (link)].
10
Genotyping of TPMT Gene Variants in IBD
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DNA of IBD patients was extracted with FALCO Biosystems. Genotyping of SNPs (rs1142345, rs1800584, rs56161402, rs6921269, rs2842951, rs2842934, rs1800460, rs2518463, rs72552739, rs1800462, and rs3898137) in the TPMT gene was performed using the Sequenom MassARRAY technology platform (Sequenom, San Diego, CA, USA), with the iPLEX Gold Reagent Kit (Sequenom), SpectroCHIP ARRAYS, Clean Resin Kit (Sequenom), and HotStarTaq DNA Polymerase (QIAGEN, Hilden, Germany) based on the single-base extension reaction. Locus-specific PCR primers and allele-specific detection primers were designed using the MassARRAY Assay Design 3.1 software (Sequenom). Allele detection was performed using a MassARRAY Analyzer Compact MALDI-TOF mass spectrometer (Sequenom), a MassARRAY Nanodispenser (Sequenom), and the MassARRAY TYPER 3.4 software (Sequenom).
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