To quantitatively evaluate the DNA methylation levels of the promoter of miR-145, genomic DNA from colonic tissues was extracted using TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) based on the manufacturer's protocols. Sequence-specific primers were designed via Agena EpiDesigner (http://www.epidesigner.org/ ) and Methprimer (http://www.urogene.org/methprimer2/tester-invitation.html ). PCR experiments were conducted, and the products were subsequently incubated with Shrimp Alkaline Phosphatase (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Promoter methylation of the miR-145 gene was measured using quantitative methylation analysis (MassARRAY Analyser; Agena Bioscience, San Diego, CA, USA), and the methylation ratios were calculated using the MassARRAY EpiTYPER software (Agena Bioscience, San Diego, CA, USA).
Massarray analyser
Manufactured by Agena
Sourced in United States
The MassARRAY analyser is a versatile laboratory instrument designed for high-throughput genetic analysis. It utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry technology to accurately detect and quantify nucleic acid sequences. The MassARRAY analyser is capable of performing a wide range of genetic assays, including genotyping, mutation detection, and gene expression analysis.
Automatically generated - may contain errors
3 protocols using massarray analyser
1
Quantitative Analysis of miR-145 Promoter Methylation
2
Genotyping of COMT polymorphism
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DNA was extracted from peripheral blood samples and stored at Karolinska Institutet Biobank. DNA samples were transferred on PCR plates and sent to the SNP&SEQ Technology Platform, Uppsala University [National Genomics Infrastructure (NGI), SciLifeLab Sweden]. The genotyping was performed using a multiplexed primer extension (SBE) chemistry of the iPLEX assay with detection of the incorporated allele by mass spectrometry with a MassARRAY analyser from Agena Bioscience (Ross et al., 1998 (link); Gabriel et al., 2009 (link); Storm et al., 2019 ). Raw data from the mass reader was converted to genotype data using the Typer software (Agena Bioscience). The COMT genotype distributions were in Hardy-Weinberg equilibrium (ps > 0.1). Of the full sample, 9 were missing genotype information and were treated as missing at random. Genotype distributions are reported in Table 1 .
3
Genotyping SLCO1B1 and SLCO1B3 Variants
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A QIAamp DNA Blood Mini Kit was used to perform genomic DNA extraction from blood samples for genotyping of SLCO1B1 (463 C>A, 388 A>G, 11187 G>A, rs4149015, 521 T>C and 1436 G>C) and SLCO1B3 (334 T>G). Genotyping was performed by multiplexed primer extension chemistry of an iPLEX assay with detection of the incorporated allele by MS with a MassARRAY analyser (Agena Bioscience, San Diego, CA, USA).17 (link),18 The output data were converted into genotype data using Typer software (Agena Bioscience).
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