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Updated: Aug 5, 2025
A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues
Published on: December 5, 2016
Rolling circle extension-assisted loop-mediated isothermal amplification (Rol-LAMP) method for locus-specific and
Jiexin Li1, Jiawang Zhou1, Yan Xia1
1School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong510006, China.
Abstract:
N6-methyladenosine (m6A) is the most prevalent RNA modification in eukaryotic mRNAs. Currently available detection methods for locus-specific m6A marks rely on RT-qPCR, radioactive methods, or high-throughput sequencing. Here, we develop a non-qPCR, ultrasensitive, isothermal, and naked-eye visible method for m6A detection based on rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP), named m6A-Rol-LAMP, to verify putative m6A sites in transcripts obtained from the high-throughput data. When padlock probes hybridize to the potential m6A sites on targets, they are converted to circular form by DNA ligase in the absence of m6A modification, while m6A modification hinders the sealing of padlock probes. Subsequently, Bst DNA polymerase-mediated RCA and LAMP allow the amplification of the circular padlock probe to achieve the locus-specific detection of m6A. Following optimization and validation, m6A-Rol-LAMP can ultra-sensitively and quantitatively determine the existence of m6A modification on a specific target site as low as 100 amol under isothermal conditions. Detections of m6A can be performed on rRNA, mRNA, lincRNA, lncRNA and pre-miRNA from biological samples with naked-eye observations after dye incubation. Together, we provide a powerful tool for locus-specific detection of m6A, which can simply, quickly, sensitively, specifically, and visually determine putative m6A modification on RNA.
Insights
We developed m6A-Rol-LAMP, a novel method for detecting N6-methyladenosine (m6A) RNA modifications. This ultrasensitive, visual technique offers a simple, rapid alternative to existing m6A detection approaches.
Area of Science:
- Molecular Biology
- Epigenetics
- Biochemistry
Background:
- N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotes, playing crucial roles in gene regulation.
- Existing methods for locus-specific m6A detection, such as RT-qPCR and sequencing, can be complex, time-consuming, or require specialized equipment.
Purpose of the Study:
- To develop a novel, ultrasensitive, isothermal, and visually detectable method for locus-specific m6A detection.
- To provide a simple, rapid, and accessible tool for verifying putative m6A sites in various RNA types.
Main Methods:
- Developed m6A-Rol-LAMP, integrating rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP).
- Utilized padlock probes that are ligated and amplified only in the absence of m6A modification, enabling indirect detection.
- Optimized the assay for sensitivity, specificity, and isothermal conditions.
Main Results:
- m6A-Rol-LAMP achieved ultra-sensitive and quantitative detection of m6A, with a limit as low as 100 amol.
- The method demonstrated naked-eye visibility for m6A detection after dye incubation.
- Successfully applied to various RNA types including rRNA, mRNA, lincRNA, lncRNA, and pre-miRNA from biological samples.
Conclusions:
- m6A-Rol-LAMP is a powerful new tool for locus-specific m6A detection.
- This method offers a simple, quick, sensitive, specific, and visual approach for identifying m6A modifications.
- Provides a valuable alternative for researchers studying RNA modifications and their functions.
