MeRIP-qPCR Assay for Detecting m6A Modification Levels of Specific RNA in Osteosarcoma Cells

Summary

This protocol provides a detailed approach to assessing m⁶A modification levels of specific RNA using MeRIP-qPCR.

Abstract

RNA epitranscriptomic modification plays a critical role in the initiation and development of various cancers, among which N⁶-methyladenosine (m⁶A) is the most prevalent and functionally diverse modification. Here, we present a streamlined and reproducible protocol using MeRIP-qPCR to assess m⁶A modification levels of specific transcripts in osteosarcoma cells. In this assay, total RNA was extracted, followed by incubation with a specific anti-m⁶A antibody to immunocapture RNA of m⁶A modification. Then, RNA was fragmented, and enrichment of m⁶A-modified RNA fragments was achieved using protein G magnetic beads. The enriched RNA was released by proteinase K digestion and purified by RNA-binding beads. Lastly, RNA was subjected to reverse transcription and quantitative polymerase chain reaction (PCR) to assess m⁶A levels in candidate genes. The results have demonstrated that this method effectively distinguishes methylation differences and provides a reliable tool for exploring the functional mechanisms of m⁶A modification in osteosarcoma cells. In conclusion, MeRIP-qPCR offers a simple and highly specific approach for studying m⁶A modification in osteosarcoma cells. This protocol holds great potential for elucidating m⁶A regulatory networks and identifying novel therapeutic targets.

Introduction

The latest epitranscriptome research reveals that RNA N⁶-methyladenosine (m⁶A) modification, the methylation of N⁶ position on adenosine, is the most abundant known intrinsic chemical modification of mRNA in eukaryotic cells¹. m⁶A is involved in a wide range of biological processes, including mRNA stability, splicing, nuclear export, and translation efficiency². Importantly, accumulating evidence suggests that dysregulation of m⁶A modification contributes to the initiation and development of various cancers, including osteosarcoma, a highly aggressive malignant bone ....

Protocol

1. Preparing the RNA

NOTE: RNA Extraction is performed using an RNA extraction kit. Work inside a clean biosafety cabinet when handling cells and RNA reagents. Wear appropriate personal protective equipment (lab coat, gloves, protective eyewear). Pre-cool the centrifuge to 4 °C before starting.

1. Prepare the cells.
   1. Remove the culture medium from the 3.5 cm dish and gently wash the cells once with pre-chilled PBS. Add an appropriate volume of RNA lysis buffer directly to the dish to fully lyse the cells and gently pipette to homogenize.
   2. Transfer the cell suspension into a 1.5 mL microcen....

Results

Here we provide a representative result of MeRIP-qPCR analysis, the m⁶A modification level of c-Myc in hFOB1.19 and 143B cells. As shown in Figure 2, c-Myc enrichment with anti-m⁶A antibody was significantly higher compared to IgG control, which only showed background levels, suggesting that the anti-m⁶A antibody is both effective and specific. We also observed that the m⁶A modification level of c-Myc was significantly higher in 143B than in hFOB1........

Discussion

MeRIP-qPCR has emerged as a valuable tool for studying RNA m⁶A modifications with broad applications. It is particularly useful in validating high-throughput sequencing results and localizing modification sites precisely within specific regions of target genes during mechanistic studies. Furthermore, this method can be readily implemented in most molecular biology laboratories due to its relatively low cost and operational simplicity, making it an efficient and practical approach for epitranscriptomic research.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100% ethanol	Shanghai Titan Scientific Co.,Ltd., America	1158566
143B	ATCC, America	ATCC CRL-8303TM
2×SYBR Green qPCR Mix	Beijing Qihengxing Biotechnology Co., Ltd., China	FS-Q1002
Denovix DS-11	Denovix	DS-11	Spectrophotometer
DMEM medium	WISENT CORPORATION, China	319-005-CL
DMEM/F-12 medium	Gibco, America	11320033
EpiQuik CUT&RUN m6A RNA Enrichment (MeRIP) Kit	Epigentek, America	P-9018-24
Fetal Bovine Serum	WISENT CORPORATION, China	086-150
hFOB1.19	Cell Bank of the Chinese Academy of Sciences, China	GNHu14
m6A RNA Methylation Assay Kit	Abcam, England	ab185912
microRNA Reverse Transcription Kit	EZBioscience-EZB, America	A0010CGQ
Real-Time System	BIO-RAD, America	CFX96
Total RNA Extraction Kit	EZBioscience-EZB, America	B0004DP
TrypLE Express Enzyme	Gibco, America	12604021