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Iplex gold assay

Manufactured by Agena
Sourced in United States

The IPLEX® Gold Assay is a versatile laboratory equipment designed for the analysis and quantification of precious metals. It utilizes advanced spectroscopic techniques to accurately measure the concentration of gold and other valuable elements in a wide range of samples. The core function of the IPLEX® Gold Assay is to provide reliable and precise analytical results for industrial, research, and academic applications.

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8 protocols using iplex gold assay

1

Genotyping of Pharmacogenetic Variants in ABC Transporters and VPA Metabolism

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Genomic DNA samples were extracted from 1.5 ml of whole blood. Target gene fragments of single nucleotide polymorphisms (SNPs) were amplified by the polymerase chain reaction (PCR), which was performed as detailed in our previous study (Fan et al., 2020 (link)). The following SNPs were selected for genotyping by reviewing the pharmacogenetic studies related to ABC transporters, VPA metabolism, nuclear receptors, and the efficacy of VPA treatment (Kwan et al., 2009 (link); El-Khodary et al., 2012 (link); Nakashima et al., 2015 (link); Queckenberg et al., 2015 (link); Li et al., 2016 (link); Talwar et al., 2017 (link); Chen et al., 2018 (link); Margari et al., 2018 (link); Wang et al., 2018 (link); Xu et al., 2018 (link); Al-Eitan et al., 2019 (link); Chouchi et al., 2019 (link); Shi et al., 2019 (link); Liu et al., 2020 (link); Makowska et al., 2021 (link)). Genotyping of all polymorphisms was carried out using Sequenom MassArray System (Agena Bioscience, San Diego, CA, United States) and iPLEX® Gold Assay. The MassArray Typer 4.0 software was used for data acquisition and analysis. All SNPs were calculated to confirm if they were in Hardy–Weinberg equilibrium.
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2

Multiplex Genotyping of FH Mutations

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The FH mutations to be studied were selected according to the known mutation frequencies from our previous study
9)
. Sequences covering the selected alterations were taken from the databases of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) and Ensembl (http://www.ensembl.org/ index.html).
The entire multiplex reaction process was performed according to the manufacturer’s instructions, including PCR amplification, shrimp alkaline phosphatase treatment, and primer extension reaction using the iPLEX Gold assay (Agena Bioscience, San Diego, California), which have been described in detail before
9)
. Briefly, genomic DNA was amplified via multiplex PCR (PCR Accessory and Enzyme Kit; Agena Bioscience) and unincorporated dNTPs were deactivated. The single-base extension reaction, consisting of the iPLEX enzyme, terminator mix, and extension primer mix, were subject to thermocycling conditions (iPLEX Gold Kit; Agena Bioscience). After desalting, the PCR products were spotted on SpectroCHIP II arrays using a MassARRAY nanodispenser and analyzed using the MassARRAY platform. Mass signals for the different alleles were captured with high accuracy by MALDI-TOF MS, and Typer v4.0 (Agena Bioscience) was used to process the raw data obtained from the assays.
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3

Peripheral Blood DNA Genotyping

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Total DNA from peripheral blood was extracted using the High Pure PCR Template Preparation kit (Roche Diagnostics GmbH, Mannheim, Germany). Next, DNA samples were genotyped at the Spanish National Genotyping Center (CeGen; http://www.cegen.org) by the Agena Bioscience’s MassARRAY platform (San Diego, CA, USA) using the iPLEX® Gold assay design system.
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4

Pharmacogenomic Genotyping of Candidate Genes

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Whole blood for DNA extraction was shipped to RUCDR Infinite Biologics (Piscataway, NJ). Purified DNA (mean concentration 10 ng/μL) was shipped to the University of Minnesota Genome Center where the Agena Bioscience iPLEX Gold assay and MassARRAY system (San Diego, CA) genotyped 8 single nucleotide variant sites across the ABCB1 (rs2032582, rs1045642, and rs1128503), CYP2B6 (rs3745274, rs2279343, and rs36079186), and NR1I3 (rs2307424 and rs3003596) genes (see supplemental materials for primer specifications). The selected variants have been associated with altered methadone or efavirenz pharmacokinetics (Bart et al., 2014 (link); Svard et al., 2010 (link)).
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5

Assessing RELN Promoter Methylation

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To assess CpG island DNA methylation, 5 mL of peripheral venous blood was taken from each participant and placed into a vacuum blood collection tube containing ethylenediaminetetraacetic acid as an anticoagulant, and was then stored at −20°C. The levels of CpG island methylation in the RELN promoter region in peripheral blood were detected using the iPLEX Gold Assay (Agena Bioscience, San Diego, USA) on the MassARRAY platform (Agena Bioscience). Samples with specific polymerase chain reaction (PCR) amplification products by both methylated and non-methylated primers were identified as positive (i.e., CpG island methylated) specimens. Only the samples of PCR products amplified by unmethylated primers were identified as negative (CpG island unmethylated). The positive rates of CpG island DNA methylation were compared between the experimental and control groups and between patients with type I and II schizophrenia.
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6

Genotyping of GABRG2 rs211037 Variant

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Genomic DNA samples were extracted from 1.5 mL of whole blood in all participants. The polymerase chain reaction was performed as described in detail in our previous study.29 (link) Genotyping of GABRG2 rs211037 was carried out using iPLEX® Gold Assay and Sequenom MassArray System (Agena Bioscience, San Diego, CA, United States). The MassArray Typer 4.0 software was used for data acquisition and analysis. The genotyping primers of GABRG2 C>T rs211037 (forward primer sequence: ACGTTGGATGTACCATCTTGGCTTCTGGTGR and reverse primer sequence: ACGTTGGATGAGCTTCTGTCTGTCAGGTCGE) were specifically synthesized in our study.
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7

CD14 rs2569190 Polymorphism Genotyping

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Genomic DNA was extracted from whole blood using High Pure PCR Template Preparation kit (Roche Diagnostics GmbH, Mannheim, Germany). CD14 rs2569190 polymorphism was genotyped at the Spanish National Genotyping Center (CeGen; http://www.cegen.org/). Genotyping was performed by Agena Bioscience’s MassARRAY platform (San Diego, CA, USA) using the iPLEX® Gold assay design system. Duplicated samples were included on each plate to check for technical replicates and negative and positive controls were used on each batch to exclude DNA contamination and ensure a technically correct laboratory process.
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8

Genotyping of Candidate SNPs

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Candidate SNPs from the exploration cohort, as well as a 7 wild-card SNPs not included on the Cardio-Metabo chip, were genotyped using the Agena Biosciences MassARRAY ® platform (formerly Sequenom). SNP multiplexes were designed using Assay Design Suite v.1.0 software (Agena Bioscience, San Diego, CA, US). Genotyping was performed according to the manufacturer's protocol using IPLEX Gold assay (Agena Bioscience, San Diego, CA, US) and analyzed using the MassARRAY Analyzer 4 platform. Mass signals for the different alleles were captured with high accuracy by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Genotype clustering and individual sample genotype calls were generated using Sequenom TyperAnalyzer v.4.0 software (Agena Bioscience, CA, US).
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