Relay-MSRE-qPCR: A sequential multi-buffer staged-dilution workflow for methylation-sensitive restriction enzyme qPCR
- 1Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University of Science, 7-Jo 15-4-1 Maeda, Teine-ku, Sapporo, Hokkaido, 006-8585, Japan
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.
Sequential staged dilution preserves enzyme-specific digestion conditions without inter-step purification.
Reduces inhibition and enhances reproducibility by budgeting carry-over components within accepted qPCR tolerance ranges.
Site-specific ΔCt shifts support reliable targeted CpG methylation analysis.
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BibTeX
@article{uemura2026relaymsreqpcr,
title = {{Relay-MSRE-qPCR: A sequential multi-buffer staged-dilution workflow for methylation-sensitive restriction enzyme qPCR}},
author = {Ippei Uemura and Takashi Satoh and Natsuko Takahashi-Suzuki},
journal = {MethodsX},
year = {2026},
month = oct,
doi = {10.1016/j.mex.2026.104205},
url = {https://doi.org/10.1016/j.mex.2026.104205},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Relay-MSRE-qPCR: A sequential multi-buffer staged-dilution workflow for methylation-sensitive restriction enzyme qPCR
AU - Ippei Uemura
AU - Takashi Satoh
AU - Natsuko Takahashi-Suzuki
PY - 2026
DA - 2026/10/12
JO - MethodsX
DO - 10.1016/j.mex.2026.104205
UR - https://doi.org/10.1016/j.mex.2026.104205
AB - 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. Sequential staged dilution preserves enzyme-specific digestion conditions without inter-step purification. Reduces inhibition and enhances reproducibility by budgeting carry-over components within accepted qPCR tolerance ranges. Site-specific ΔCt shifts support reliable targeted CpG methylation analysis.
SN - 2215-0161
ER -