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Multiplexed and Sensitive DNA Methylation Testing Using Methylation-Sensitive Restriction Enzymes "MSRE-qPCR".
Gabriel Beikircher1, Walter Pulverer1, Manuela Hofner1
1Molecular Diagnostics Unit, AIT-Austrian Institute of Technology GmbH, Muthgasse 11, Vienna, 1190, Austria.
Methods in Molecular Biology (Clifton, N.J.)
|December 11, 2017
Summary
This study introduces a new method, MSRE-qPCR, for efficiently confirming DNA methylation biomarkers. It enables quantitative, multiplexed analysis of small DNA amounts, crucial for cancer epigenetics and liquid biopsies.
Area of Science:
- Epigenetics and Molecular Genetics
- Cancer Research and Diagnostics
Background:
- DNA methylation, specifically 5-methylcytosine (5mC) in CpG contexts, is a stable epigenetic marker crucial in gene regulation.
- Traditional methods like methylation-sensitive restriction enzymes (MSRE) and bisulfite sequencing have limitations in validating numerous DNA methylation biomarkers, especially with limited DNA samples.
- Analyzing cell-free DNA (cfDNA) and circulating tumor cells (CTCs) for disease-specific methylation patterns presents challenges in confirming candidate biomarkers.
Purpose of the Study:
- To develop and present a novel method, MSRE-qPCR, for efficient and quantitative confirmation of DNA methylation.
- To address the bottleneck in validating multiple DNA methylation biomarker candidates from discovery studies.
- To enable sensitive detection of DNA methylation in low-input samples, including cfDNA and CTCs.
Main Methods:
- Development of a Methylation-Sensitive Restriction Enzyme quantitative Polymerase Chain Reaction (MSRE-qPCR) assay.
- Application of MSRE-qPCR for quantitative, multiplexed (48-96 plex) DNA methylation analysis.
- Adaptation of the method for both standard and high-throughput nanoliter-scaled qPCR settings.
Main Results:
- MSRE-qPCR allows efficient confirmation and testing of DNA methylation in a quantitative multiplexed manner.
- The method is suitable for analyzing very small amounts of DNA (nanogram quantities).
- It enables sensitive detection of low percentages (0.1-1%) of specific methylated DNA in an unmethylated background, detecting fewer than 10 target copies.
Conclusions:
- MSRE-qPCR offers a powerful solution for validating DNA methylation biomarkers, overcoming limitations of previous techniques.
- The method is highly applicable to clinical research and diagnostics, particularly for analyzing cfDNA and CTCs in liquid biopsies.
- This approach facilitates the translation of epigenome-wide discovery findings into validated diagnostic or prognostic markers.
