Of the 150 EOC tissue samples, 40 yielded high-quality DNA using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. MALDI-TOF mass spectrometry (Sequenom, San Diego, California, USA), described by Breitling et al. [31 (link)], was performed for hMSH2 methylation analysis by CapitalBio Co., Ltd. (Beijing, China).
Maldi tof mass spectrometry
Manufactured by Labcorp
Sourced in United States, Netherlands
MALDI-TOF mass spectrometry is an analytical technique used for the identification and characterization of biomolecules. It employs matrix-assisted laser desorption/ionization (MALDI) to ionize samples, which are then analyzed by a time-of-flight (TOF) mass spectrometer. This method enables the determination of the molecular masses of a wide range of molecules, including proteins, peptides, oligosaccharides, and other biomolecules.
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Lab products found in correlation
Wizard genomic dna purification kit, by Promega (2 mentions)
Massarray system, by Labcorp (2 mentions)
Epityper software, by Labcorp (2 mentions)
Iplex gold technology, by Labcorp (1 mentions)
Blood genome dna extraction kit, by Takara Bio (1 mentions)
Peak scanner software, by Thermo Fisher Scientific (1 mentions)
Snapshot, by Thermo Fisher Scientific (1 mentions)
Epityper software v1, by Labcorp (1 mentions)
Massarray compact maldi tof, by Labcorp (1 mentions)
Spectrochip array, by Labcorp (1 mentions)
Massarray iplex gold system, by Labcorp (1 mentions)
Ez 96 dna methylation gold kit, by Zymo Research (1 mentions)
Massarray, by Labcorp (1 mentions)
Iplex technology, by Labcorp (1 mentions)
Massarray typer software, by Labcorp (1 mentions)
Massarray nanodispenser rs1000, by Labcorp (1 mentions)
15 protocols using maldi tof mass spectrometry
1
DNA Extraction and Methylation Analysis
2
p53 Genotyping by MALDI-TOF Mass Spectrometry
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Genomic DNA was extracted from peripheral blood lymphocytes of patients. Genotyping was performed using MassARRAY high-throughput DNA analysis with Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Sequenom, Inc., San Diego, CA). SNP (G-to-C transversion) at codon 72 (rs1042522) of the gene encoding p53 (TP53) was genotyped using iPLEX Gold technology following manufacturer protocol (Sequenom). Detailed description of methods has been previously reported [65 (link)].
3
Genotyping of NBS1 SNPs
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Based on the possibility of functional change of NBS1 SNPs in the genome context, four NBS1 SNPs (924T/C in 5’-UTR, rs1805794G/C in exon 5, 31129G/A in exon 10 and rs2735383C/G in 3’-UTR) previously reported in relation to disease outcomes including cancers were selected and analyzed [22 (link), 25 (link)]. Genomic DNA was isolated from peripheral blood samples for each study subject using the Blood Genome DNA Extraction Kit (TaKaRa; Dalian, China). These four NBS1 SNPs were genotyped by allele-specific MALDI-TOF mass spectrometry (Sequenom, San Diego, CA) as described before[26 (link), 27 (link)] without the knowledge of the subjects status. Also, to confirm the genotyping results from the mass spectrometric analysis, we randomly selected 100 samples for direct sequencing, and the result was 100% concordant.
4
Whole Blood Genomic DNA Extraction
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Peripheral whole blood samples were obtained from all the participants by venepuncture, and genomic DNA was prepared using a modified salting-out procedure [36] (link). Prioritized SNPs were genotyped by primer extension reaction followed by the MALDI-TOF mass spectrometry (Sequenom). Automated calls were made using Mass Array Typer module of the software. To rule out any genotyping error, 5% of the total samples were re-genotyped randomly using single-base primer extension methods (SNaPshot, Applied Biosystems). Calls were made after analyzing data in PeakScanner Software (Applied Biosystems). Allele calls, clustering patterns and peak intensities were reviewed by an experienced technician. The SNPs with call rate exceeding 98% were considered.
5
Quantifying DNA Methylation in High-Grade Serous Ovarian Cancer
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Of the 96 HGSOC samples, high-quality DNA from 26 HGSOC tissue samples was isolated using the Wizard Genomic DNA Purification Kit (Promega, Madison, Wisconsin), as described by the manufacturers. MALDI-TOF mass spectrometry (Sequenom, San Diego, California, U.S.) was used to detect the methylation level of the MGRN1 promoter region. This experiment was conducted at CapitalBio Co., Ltd. (Beijing, China). PCR primers were designed using Methprimer (http://www.urogene.org/methprimer ). For each reverse primer, an additional T7 promoter tag for in vivo transcription was added, whereas a 10 m tag on the forward primer was used to adjust melting temperature differences. Mass spectra were obtained via MassARRAY Compact MALDI-TOF (Sequenom). The resultant methylation calls were analysed with EpiTyper software v1.0 (Sequenom) to generate quantitative results for each CpG site or an aggregate of multiple CpG sites.
6
Genotyping Protocol for Genetic Association
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The selected SNPs were genotyped on Sequenom MassARRAY system.21 Briefly, gDNA samples were diluted to approximately 5‐10 ng/μL and amplified by multiplex PCR. PCR products were applied for locus‐specific single‐base extension reactions. Resulting products were desalted and transferred to a 384‐element SpectroCHIP array for the allele detection on MALDI‐TOF mass spectrometry (Sequenom).
The associations were analyzed by comparing MAF in cases with those of controls using PLINK 1.07 software,22 using logistic regressions with an additive model to obtain the P values, odds ratios, and 95% confidence interval. Age and gender were included as covariates. All SNPs should pass the quality control (call rate > 95%, Hardy‐Weinberg equilibrium P > .001, in the controls). The genetic statistical power was estimated for all genotyped SNPs using CaTS‐Power Calculator software.23
The associations were analyzed by comparing MAF in cases with those of controls using PLINK 1.07 software,
7
Genotyping of miR-1658 SNP in F2 Chickens
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The rs16681031 SNP occurring in miR-1658 (+60 bp C>G) in the F2 chickens was genotyped using MassArray-iPLEX GOLD System (Sequenom Inc., USA). The SNP genotyping was accomplished with a pair of sequence amplification primers and a single-base extension primer designed using Sequenom software Assay Design 3.1 version. The first PCR primer sequence of pre-mir-1658 SNP was 5′-ACGTTGGATGTTCTGCCATAC CAGTGTGTG -3′; the second PCR primer sequence was 5′-ACGTTGGATGACTCCTCCAGCTGCTGCTTC -3′, and the single-base extension primer was 5′-CATCAACACCAACCCA -3′. The SNP of pre-mir-1658 was genotyped using Sequenom MALDI-TOF mass spectrometry according to the manufacturer’s instructions [28 ]. Related genotyping data including the peak area and call rate were obtained. Alleles were assigned automatically, using the software package provided by the manufacturer.
8
MALDI-TOF Mass Spectrometry for DNA Methylation
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MALDI-TOF mass spectrometry (Sequenom) described by Breitling et al. [9 (link), 41 (link)] was used in all the validation and further exploring rounds. In brief, DNA was bisulfite converted by EZ-96 DNA Methylation Gold Kit (Zymo Research) and amplified by bisulfite-specific primers (no SNPs in the primers) and PCR amplicons HYAL2-A were described as previously used [25 (link)]. The PCR products were used according to the standard protocol of Sequenom EpiTyper Assay, and further cleaned by Resin and dispensed to a 384 SpectroCHIP by a Nanodispenser as described by us before. The chips were read by a Sequenom Mass Spectrometer system. Data were collected by SpectroACQUIRE v3.3.1.3 software and visualized with MassArray EpiTyper v1.2 software. For each batch of MassARRAY analysis, same amount of cases and controls were randomly selected from the cohort [25 (link)].
9
Genotyping of ITPK1 Gene Variants
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All cases and controls were genotyped for the selected SNPs of the ITPK1 gene were using Sequenom MassARRAY System with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Sequenom, San Diego, California). Two primers for Polymerase chain reaction (PCR) amplification and one primer for extension primers were designed using RealSNP (https://www.mysequenom.com/ ). Approximately 20 ng of genomic DNA was used to genotype each sample. The data were analyzed using Type Analysis 4.0. We had done the validation study about the genotyping accuracy in ITPK1 gene. The Sequenom genotyping platform is medium-throughput. In order to ensure the genotyping consistency, 10% of samples were re-genotyped, and another 10% of samples were sequenced directly to validate the accuracy of the genotyping. Sequencing results were exported to Mutation Surveyor Version 3.25 (Softgenetics, State College, PA, USA; http://www.softgenetics.com ) for alignment and multiple comparisons. Genotyping quality control study contained 1 blank sample, 2 repeated samples, and a >90% of genotyping call rate.
10
Quantitative DNA Methylation Analysis
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The Sequenom MassARRAY platform (San Diego, USA) was used to perform the quantitative methylation analysis of imprinted genes. This system, which combines base-specific enzymatic cleavage with matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, is a highly accurate, sensitive and high-throughput method for the quantitative analysis of DNA methylation at CpG sites.18 (link) DNA methylation at selected imprinted gene DMRs of four imprinted genes were quantified with the Sequenom MassARRAY EpiTYPER analyzer. The robustness of this approach for quantifying methylated/unmethylated DNA has been demonstrated by Sequenom. Primers used in this study were designed by using Methprimer (http://epidesigner.com ). The primer sequences and methods of the imprinted genes are mentioned in our previous study.17 (link) The spectra and the methylation values of MALDI-TOF mass spectrometry (Sequenom) were collected and analyzed by using Epityper software (version 1.0; Sequenom). The DNA methylation assays were performed in triplicate. Inapplicable readings and their corresponding sites were eliminated from analysis. The average methylation was calculated as the mean value of the CpGs methylation rate and expressed as a relative amount of methylation.
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