{
  "abstract": "Methylation-sensitive restriction enzyme quantitative PCR (MSRE-qPCR) quantifies locus-specific DNA methylation in native DNA without bisulfite conversion. However, such multi-enzyme workflows are limited by buffer incompatibility, cumulative inhibitor carry-over, and sample loss during purification. We developed Relay-MSRE-qPCR, a sequential staged-dilution workflow in which each heat-inactivated digest is transferred to a fresh tube, diluted ≥5-fold to attenuate inhibitors, and then the buffer for the next enzyme is reconstituted to 1 × concentration. Buffer budgeting kept carry-over compatible with qPCR, with ≤5% (v/v) glycerol included per step. With Eco52I (XmaIII), HhaI, EcoRII, VpaK11BI (AvaII), and HpaII, the relay maintained restriction-site-responsive digestion and the cumulative amount of salts, Mg²⁺, and stabilizers remained within polymerase-tolerable limits; defined unmethylated PCR-product controls showed marked loss of amplifiable template for all five enzymes. Relay and single-enzyme comparator conditions showed consistent delta cycle threshold (ΔCt) behavior, and qPCR products exhibited single-peak melt curves under the tested conditions. Only enzymes recognizing a site within the PCR amplicon produced ΔCt shifts, confirming site-specific responsiveness. A practical protocol is provided with guidance for amplicon placement, enzyme selection, and buffer accounting. Relay-MSRE-qPCR enables reproducible, low-cost targeted CpG methylation analysis when base-resolution sequencing is unnecessary or impractical.\n\nSequential staged dilution preserves enzyme-specific digestion conditions without inter-step purification.\n\nReduces inhibition and enhances reproducibility by budgeting carry-over components within accepted qPCR tolerance ranges.\n\nSite-specific ΔCt shifts support reliable targeted CpG methylation analysis.",
  "abstract_shown": true,
  "affiliations": [
    "Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University of Science, 7-Jo 15-4-1 Maeda, Teine-ku, Sapporo, Hokkaido, 006-8585, Japan"
  ],
  "article_type": "research article",
  "arxiv": null,
  "author_total": 3,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Ippei Uemura"
    },
    {
      "affs": [
        0
      ],
      "name": "Takashi Satoh"
    },
    {
      "affs": [
        0
      ],
      "name": "Natsuko Takahashi-Suzuki"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/817ghc5f5f9c8.bib",
  "cited_by": 0,
  "doi": "10.1016/j.mex.2026.104205",
  "fulltext_url": "https://pmc.ncbi.nlm.nih.gov/articles/PMC13734947/",
  "id": "817ghc5f5f9c8",
  "issns": [
    "2215-0161"
  ],
  "landing_url": "https://doi.org/10.1016/j.mex.2026.104205",
  "language": "en",
  "licence": "CC-BY-4.0",
  "licence_label": "CC BY 4.0",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "heading",
      "Biochemistry, Genetics and Molecular Biology"
    ],
    [
      "openalex",
      "1312|Epigenetics and DNA Methylation"
    ]
  ],
  "openalex": "W7221567905",
  "pdf_url": null,
  "pmcid": "PMC13734947",
  "pmid": "42990954",
  "published": "2026-10-12",
  "publisher": "Elsevier",
  "ris": "https://research.draconitic.com/paper/817ghc5f5f9c8.ris",
  "sources": [
    {
      "first_seen": "2026-11-09T12:20:44Z",
      "native_id": "PMC13734947",
      "source": "pmc",
      "url": "https://doi.org/10.1016/j.mex.2026.104205"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1312|Epigenetics and DNA Methylation",
      "subfield": "molecular-biology"
    }
  ],
  "title": "Relay-MSRE-qPCR: A sequential multi-buffer staged-dilution workflow for methylation-sensitive restriction enzyme qPCR",
  "title_text": "Relay-MSRE-qPCR: A sequential multi-buffer staged-dilution workflow for methylation-sensitive restriction enzyme qPCR",
  "type": "peer-reviewed",
  "updated_at": "2026-11-09T12:20:44Z",
  "url": "https://research.draconitic.com/paper/817ghc5f5f9c8",
  "venue": "MethodsX",
  "versions": []
}