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RNA-seq

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...

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Updated: Nov 1, 2025

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

Published on: December 5, 2016

Mapping single-nucleotide m⁶A by m⁶A-REF-seq.

Hong-Xuan Chen¹, Zhang Zhang¹, Dong-Zhao Ma¹

¹MOE Key Laboratory of Gene Function and Regulation, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.

Methods (San Diego, Calif.)

|June 26, 2021

Summary

This summary is machine-generated.

Researchers developed m6A-REF-seq, a new method to precisely map N6-methyladenosine (m6A) RNA modifications. This technique overcomes limitations of existing methods, enabling better functional studies of m6A in cells.

Keywords:

Epitranscriptome RNA modification Single-base resolution m(6)A

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Area of Science:

Molecular Biology
Epigenetics
Genomics

Background:

RNA modifications, particularly N6-methyladenosine (m6A), are crucial for eukaryotic mRNA regulation and cellular functions.
Current m6A detection methods have limitations, hindering comprehensive functional studies.
Existing techniques often rely on m6A antibodies, which can introduce biases.

Purpose of the Study:

To introduce m6A-REF-seq, a novel, straightforward, and high-resolution method for identifying m6A modifications.
To provide an orthogonal approach to m6A detection, independent of antibody-based methods.
To detail a protocol for m6A-REF-seq, including library preparation and data analysis.

Main Methods:

m6A-REF-seq utilizes the RNA endonuclease MazF, which specifically recognizes and cleaves at the m6A-containing ACA motif.
The method involves next-generation sequencing (NGS) library construction and subsequent bioinformatic analysis of sequencing data.
An optimized assay is described for validating individual m6A sites identified by m6A-REF-seq.

Main Results:

m6A-REF-seq enables the identification of m6A modifications at single-nucleotide resolution.
The method provides an antibody-independent approach, offering an orthogonal validation strategy.
The protocol is detailed, facilitating its implementation in other research settings.

Conclusions:

m6A-REF-seq is a powerful tool for advancing the study of m6A RNA modifications.
This method overcomes previous technical limitations, paving the way for deeper functional investigations of m6A.
The developed assay can be broadly applied for validating candidate m6A sites.