After centrifugation, blood samples were placed in -80℃ refrigerators. We extracted and concentrated the genomic DNA from samples according to the methods in our previous studies 22 (link), 23 (link). We selected rs1545224 and rs2241883 polymorphisms to explore the association, genotyped them by the Sequenom MassARRAY RS1000 and analyzed the data by Sequenom Type 4.0. Primers used are shown in Table 1 .
Massarray rs1000
Manufactured by Labcorp
Sourced in United States
The MassARRAY RS1000 is a laboratory instrument that uses a mass spectrometry-based technology to analyze genetic variations. It is designed to perform high-throughput genotyping and gene expression analysis. The core function of the MassARRAY RS1000 is to detect and measure specific DNA or RNA molecules in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Typer 4, by Labcorp (24 mentions)
Massarray assay design 3, by Labcorp (21 mentions)
Du530 uv vis spectrophotometer, by Beckman Coulter (19 mentions)
Sequenom massarray assay design 3, by Labcorp (8 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Sequenom typer 4, by Labcorp (3 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (3 mentions)
Sequenom typer 3, by Labcorp (3 mentions)
Nanodrop, by Thermo Fisher Scientific (3 mentions)
Typer 3, by Labcorp (3 mentions)
Universal genomic dna extraction kit, by Takara Bio (2 mentions)
Purelink pro 96 genomic dna purification kit, by Thermo Fisher Scientific (2 mentions)
Clean resin, by Labcorp (1 mentions)
Gold mag mini whole blood genomic dna purification kit, by Takara Bio (1 mentions)
Taq dna polymerase, by Solgent (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
Nanodrop 2000 device, by Thermo Fisher Scientific (1 mentions)
Blood dna extraction kit, by Tiangen Biotech (1 mentions)
Abi prism 3730xl dna sequencer, by Thermo Fisher Scientific (1 mentions)
51 protocols using massarray rs1000
1
Genomic DNA Extraction and Genotyping
2
Genomic DNA Extraction and Genotyping
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We collected these blood samples from each individual after their admission to the hospital. The whole blood samples of all participants collected in a test tube containing EDTA were used for genotyping assay. Genomic DNA was isolated from peripheral white blood and concentrated by using GoldMag-Mini Whole Blood Genomic DNA Purification Kit according to the manufacturer's directions (GoldMag Co. Ltd. Xian, China). The extracted DNA was stored at -80℃ until use. We adopted the spectrometry (DU530 UV/vis spectrophotometer, Beckman Instruments, Fullerton, CA, USA) to detect DNA purity. Sequenom MassARRAY Assay Design 3.0 Software was used to design Multiplexed SNP MassEXTEND assay 19 . PCR and extension primers were designed by Sequenom, Inc. Assay Design. EXO-SAP was used to digest PCR-amplified DNA, and then mixed the primer extended by IPLEX chemistry, desalted using Clean Resin (Sequenom) and spotted onto Spectrochip matrix chips. Finally, results were detected by Mass Spectrometer. All samples were genotyped by Sequenom MassARRAY RS1000 according to the manufacturer's protocol. The final data was managed and analysed by Sequenom Typer 4.0 Software 19 , 20 (link).
3
Genotyping of Blood Leukocyte DNA SNPs
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Peripheral blood samples were collected in an anticoagulation tube and stored at –80°C until detection before subjects had received other therapies. Depend on manufacturer′s instructions of the GoldMag-Mini Purification Kit (GoldMag Co.Ltd. Xi′an city, China), genomic DNA was isolated from blood leukocytes samples. At the same time, the concentrations and purity of the DNA were measured by using the NanoDrop 2000 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at a wavelength of A260 and A280 nm.
A total fourteen SNPs were selected at a minor allele frequency > 5% in the HapMap Chinese Han Beijing (CHB) population. Sequenom Mass-ARRAY RS1000 (Sequenom, San Diego, CA) was used to genotype the SNPs rs6713088, rs12621038, rs1682111, rs843752, rs10439478, rs843645, rs11125529, rs12615793, rs843711, rs11896604, rs843706, rs17045754, and rs843720. Data management and analysis were managed by using Sequenom Typer 4.0 Software (Sequenom Co. Ltd) [25 (link), 26 (link)]. The PCR primers for the fourteen selected tSNPs were shown in Table6 .
A total fourteen SNPs were selected at a minor allele frequency > 5% in the HapMap Chinese Han Beijing (CHB) population. Sequenom Mass-ARRAY RS1000 (Sequenom, San Diego, CA) was used to genotype the SNPs rs6713088, rs12621038, rs1682111, rs843752, rs10439478, rs843645, rs11125529, rs12615793, rs843711, rs11896604, rs843706, rs17045754, and rs843720. Data management and analysis were managed by using Sequenom Typer 4.0 Software (Sequenom Co. Ltd) [25 (link), 26 (link)]. The PCR primers for the fourteen selected tSNPs were shown in Table
4
Genotyping Common ACYP2 Variants
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We randomly selected seven potentially function SNPs in the ACYP2 gene (rs6713088, rs12621038, rs1682111, rs843752, rs10439478, rs17045754, and rs843720) for analysis. The SNPs were selected based on population and MAF > 5% using dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP ). These SNPs represented the majority of known common variants in ACYP2. Genotyping was performed using a Sequenom MassARRAY RS1000 (Sequenom, Inc., San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, locus-specific polymerase chain reaction (PCR) amplification was performed and the products purified by addition of shrimp alkaline phosphatase. Single base extension was then performed using primers that annealed immediately upstream of each SNP. Finally, the alleles were determined by mass spectrometry of the extended primers. The primers used for each SNP are listed in Table 7 .
5
Genetic Variant Identification Protocol
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We selected genetic variants from published polymorphisms associated with VIP variants from the PharmGKB database. We designed assays for the 85 genetically-variant loci in 37 genes that formed the basis for our our analyses. We excluded loci if we could not design an assay. We extracted genomic DNA from peripheral blood obtained from the subjects using the GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMagLtd. Xi’an, China) according to the manufacturer’s protocol. The DNA concentration was measured with a NanoDrop 2000C spectrophotometer (Thermo Scientific, Waltham, MA, USA). The Sequenom MassARRAY Assay Design 3.0 software (San Diego, CA, USA) was used to design multiplexed single nucleotide polymorphism (SNP) MassEXTEND assays [6 ]. SNP genotyping analysis was performed using the standard protocol recommended by the manufacturer with a Sequenom MassARRAY RS1000. Sequenom Typer 4.0 software was used to manage and analyze the SNP genotyping data as described in a previous report [7 (link)].
6
Genotyping of TIMP-2 Gene Variants
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Candidate SNPs in the TIMP-2 gene from the databases of 1000 Genomes Project (http://www.1000genomes.org/ ) and dbSNP (https://www.ncbi.nlm.nih.gov/projects/SNP/ ) with MAFs >5% in Chinese Han Beijing (CHB) population, and had some previous studies were chosen. A total of six SNPs (i.e., rs2277698, rs2009196, rs7342880, rs11654470, rs2003241, rs4789936) were selected for further genotyping. These SNPs were analyzed in vascular diseases or other diseases, such as the breast cancer [25 (link)], abdominal aortic aneurysm [26 (link)], knee osteoarthritis [31 ]. Genomic DNA was extracted from peripheral blood of cases and controls using the GoldMag whole blood genomic DNA purification kit (GoldMag Co. Ltd., Xi’an, China), as recommended by the manufacturer's instructions [40 (link)]. DNA concentration was determined by the NanoDrop 2000C spectrophotometer (Thermo Scientific, Waltham, MA, USA). Sequenom MassARRAY Assay Design 3.0 software (San Diego, CA, USA) was used to design primers for amplification process and single base extension reactions [41 (link)]. SNP genotyping was carried out by Sequenom MassARRAY RS1000 (Sequenom, SanDiego, CA). Sequenom Typer 4.0 software was used to manage and analyze SNP genotypic data.
7
Genetic Variants Associated with Urinary Cancers
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We reviewed the literatures related to association between TERT and TERC polymorphisms and tumors of urinary system and selected SNPs in TERT and TERC with the minor allele frequencies (MAF) ≥5% in Asian by using HapMap database [22 (link)–24 (link), 38 ]. In addition, the correlation between chosen SNPs and RCC in Chinese Han population has not been reported before. Genomic DNA was extracted from whole blood samples using the Gold Mag-Mini Whole Blood Genomic DNA Purification Kit (version 3.0; TaKaRa, Japan) [39 ]. The DNA concentration was measured by spectrometry (DU530 UV/VIS spectrophotometer, Beckman Instruments, Fullerton, CA, USA). The Sequenom MassARRAY Assay Design 3.0 software (Sequenom, Inc, San Diego, CA, USA) was used to design the multiplexed SNP Mass EXTEND assay. Genotyping was performed using a Sequenom MassARRAY RS1000 (Sequenom, Inc.) in accordance with the manufacturer's protocol. Sequenom Typer 4.0 software was used to perform data management and analyses [40 ]. The primers corresponding to each SNP are listed in Table 7 . Based on these results, six SNPs including rs35073794, rs10936599, rs10069690, rs2242652, rs2853677, rs2853676 were selected.
8
Genetic Polymorphisms Genotyping Using Sequenom MassARRAY
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Using the PharmGKB database, we screened published genetic polymorphisms associated with VIP variants, and finally 85 genetic variant loci from 37 genes were randomly selected for our investigation. We extracted genomic DNA from whole blood using a GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMag Ltd. Xi’an, China) according to the manufacturer’s protocol. The genomic DNA concentration was measured by absorbance at 260 nm using a NanoDrop 2000C (Thermo Scientific, Waltham, Massachusetts, USA). We used the Sequenom MassARRAY Assay Design 3.0 software (San Diego, California, USA) to design multiplexed SNP MassEXTEND arrays [12 ]. We utilized a Sequenom MassARRAY RS1000 (San Diego, California, USA) to genotype the SNPs according to the manufacturer’s instructions. Sequenom Typer 4.0 Software was used for data collection and analysis as described previously [13 (link)].
9
Genotyping of TIM Gene SNPs
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Whole blood samples were placed into EDTA-coated tubes and preserved at −80°C until further use. Genomic DNA was isolated from the whole blood samples using a standard phenol-chloroform extraction method. DNA concentrations were measured via spectrometry (DU530 UV/VIS spectrophotometer; Beckman Instruments, Fullerton, CA, USA). For our study, we selected candidate SNPs in TIM according to HapMap data from a Chinese population. To achieve a power of at least 50%, only SNPs with a minor allele frequency (MAF) > 0.01 was included. Sequenom MassARRAY Assay Design 3.0 Software (Agena Bioscience, San Diego, CA, USA) was used to design a Multiplexed SNP MassEXTEND assay. Finally, three SNPs (rs2229080, rs7504990, and rs4078288) were selected according to Chinese population data from HapMap. DCC SNP genotyping was performed using a Sequenom MassARRAY RS1000, according to the manufacturer's instructions. The corresponding primers used for each SNP in this study are listed in Table 6 . Sequenom Typer 3.0 Software (Sequenom Inc., San Diego, CA, USA) was used for data analysis.
10
Identifying Tagging SNPs in EGFR for Glioma Research
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Using the public dbSNP (https://www.ncbi.nlm.nih.gov/snp ) and HapMap (http://www.hapmap.org/ ) and identify linkage disequilibrium (LD) blocks within EGFR to get a dense tagging of SNPs, we used Haploview software (http://www.broad.mit.edu/mpg/haploview/ ) setting the minimum r2 to 0.9 and the minimum minor allele frequency to 5 % in HapMap Chinese Han Beijing residents. Finally we selected candidate 8 tSNPs in the EGFR gene for genotyping, which previously reported being associated with gliomas [16 (link)]. Genomic DNA was extracted from whole blood using the GoldMag® nanoparticles method (GoldMag Ltd. Xi’an, China) according to the manufacturer's instructions, and the DNA concentration was measured with the NanoDrop 2000C (Thermo Scientific, Waltham, Massachusetts, USA). The Sequenom MassARRAY Assay Design 3.0 Software (San Diego, California, USA) was used to design Multiplexed SNP MassEXTEND assays [17 (link)]. SNPs genotyping was performed by the Sequenom MassARRAY RS1000 (San Diego, California, USA) and Sequenom Typer 4.0 Software (San Diego, California, USA) was used to perform data management and analysis as previously described [17 (link), 18 (link)].
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