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 tumor that predominantly affects adolescents and young adults³.

The study of m⁶A modification requires robust and reliable methodologies. Several approaches have been developed to detect m⁶A modification levels, including m⁶A dot blot, m⁶A ELISA, and MeRIP-seq. The m⁶A dot blot assay is simple and cost-effective, yet it suffers from limited sensitivity and poor quantification. It provides the m⁶A modification level of total RNA, rather than the m⁶A modification level of a specific RNA⁴. m⁶A ELISA offers relatively convenient and high-throughput detection; however, cross-reactivity and low accuracy remain major concerns, and it similarly lacks the capacity to reveal transcript- or site-specific modifications⁵. MeRIP-seq enables transcriptome-wide mapping of m⁶A; however, its requirements for large RNA input, relatively low resolution, and antibody dependency limit MeRIP-seq precision and reproducibility⁶. By contrast, MeRIP-qPCR combines immunoprecipitation with quantitative PCR, providing higher sensitivity and specificity for targeted detection of m⁶A on specific RNA⁷. This method enables sensitive and targeted detection of differential m6A enrichment in candidate genes, making it particularly suitable for validation studies in m6A-related cancer research. This method offers a cost-effective and accessible alternative for laboratories aiming to investigate m6A-mediated regulatory mechanisms.

Here, we provided a detailed protocol (Figure 1) to assess m⁶A modification levels of c-Myc in osteosarcoma cells using MeRIP-qPCR as an example. MeRIP-qPCR, relying on antibody-based enrichment of methylated RNA fragments, also coupled with reverse transcription and quantitative PCR, evaluates the presence and relative abundance of m⁶A on specific RNA. We demonstrated that this approach effectively distinguished methylation differences of c-Myc and provided a reliable platform for investigating the functional role of m⁶A in osteosarcoma cells. This study highlights the potential of MeRIP-qPCR as a practical and specific tool for m⁶A modification research in osteosarcoma and sets the stage for identifying novel therapeutic targets.

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 microcentrifuge tube.
   3. Centrifuge at 1000 × g for 5 min at 4 °C.
      NOTE: Ensure that the rotor is balanced and the lid is closed securely.
   4. Carefully take out the microcentrifuge tube after centrifugation.
   5. Aspirate the supernatant completely using a 1 mL pipette without disturbing the pellet at the bottom.
      NOTE: Do not touch the cell pellet with the pipette tip.
2. Lyse cells.
   1. Add 300 µL of Lysis Buffer to the cell pellet.
   2. Mix thoroughly by inverting the tube 30 times until the solution appears clear and viscous with no visible clumps.
   3. Incubate the tube at room temperature for 2 min.
3. Precipitate RNA.
   1. Add 300 µL of 100% ethanol to the lysate (equal volume of Lysis Buffer).
   2. Gently pipette up and down immediately (about 8-10 times) to fully mix.
4. Perform RNA column binding.
   1. Place an RNA purification column into a provided 2 mL collection tube.
   2. Transfer the entire mixture to the purification column.
      NOTE: Avoid touching the white membrane in the center.
   3. Close the cap and centrifuge at 12,000 × g for 1 min.
   4. Discard the lower liquid and gently tap the collection tube upside down on absorbent paper to remove residual liquid.
   5. Return the purification column to the collection tube.
5. Wash impurities.
   1. Add 500 µL of Wash Buffer ( Nuclease-free water) to the purification column.
      NOTE: Avoid touching the white membrane in the center.
   2. Centrifuge at 12,000 × g for 1 min and discard the lower liquid.
   3. Centrifuge again at 12,000 × g for 1 min to remove residual liquid.
   4. Transfer the purification column into a clean 1.5 mL microcentrifuge tube.
   5. Open the cap and let the column stand for 2 min at room temperature.
6. Elute RNA.
   1. Place the purification column into a new 1.5 mL microcentrifuge tube.
   2. Add 25 µL of Elution Buffer directly onto the center of the membrane.
   3. Incubate at room temperature for 5 min.
   4. Centrifuge at 12,000 × g for 1 min to collect RNA in the lower tube.
   5. Reapply the eluate to the membrane and centrifuge again at 12,000 × g for 1 min to maximize the yield.
      NOTE: Always add elution buffer directly onto the center of the column membrane. Avoid adding it to the side wall, as this may prevent RNA from being dissolved and greatly reduce the yield. Also, do not puncture the membrane with the pipette tip.
7. Measure RNA concentration.
   1. Rinse the spectrophotometer sample port with 1.5 µL of Elution Buffer.
   2. Use 1.5 µL of Elution Buffer as a blank. Confirm that the reading is close to zero.
   3. Load 1.5 µL of RNA sample and measure the concentration.
   4. Assess the quality of RNA samples using a spectrophotometer (A260/A280 = 1.8-2.2, A260/A230 = 2.0-2.5) and agarose gel electrophoresis.

2. Methylated RNA immunoprecipitation (MeRIP)

1. Preparation of immunocapture mixture
   1. Obtain a new 0.2 mL PCR tube.
   2. Add components as listed in Table 1 (example for one reaction).
2. Immunocapture
   1. Close the tube cap tightly. Place the PCR tube on a rotator or rolling shaker.
   2. Incubate at room temperature for 90 min.
      NOTE: Ensure that the solution is mixed continuously and gently.
3. RNA cleavage
   1. After incubation, add 10 µL of Nuclear Digestion Enhancer (NDE) and 2 µL of Cleavage Enzyme Mix (CEM) to each tube.
   2. Mix gently by pipetting up and down 5 to 6 times.
   3. Incubate at room temperature for 4 min.
   4. Preparation of the Input RNA control
      1. To ensure consistency between immunoprecipitation (IP) and Input samples, process a portion of RNA in parallel as the Input control.
      2. Transfer 1 µg of total RNA (10% input) from each sample into a separate PCR tube. Add 20 µL of immuno capture buffer (ICB), 1.5 µL of NDE, 1 µL of CEM, and incubate at room temperature for 2 min.
4. Magnetic enrichment and washing
   1. Place the PCR tube on a magnetic stand.
   2. Allow the tube to sit for 2 min until the solution becomes clear and the beads are captured on the tube wall.
   3. Carefully aspirate and discard the supernatant without disturbing the beads.
   4. Keep the tube on a magnetic stand and add 150 µL of Wash Buffer (WB).
   5. Remove the tube from the magnetic stand and tilt it upside down gently to fully resuspend the beads.
   6. Return the tube to the magnetic stand. Wait 1-2 min until the solution clears, then discard the supernatant.
   7. Repeat the wash step three times.
   8. Wash the beads once more with 150 µL of Protein Digestion Buffer (PDB) using the same method.
5. Release and recovery of enriched RNA
   1. Prepare Protein Digestion Working Solution by mixing Proteinase K and PDB in a 1:9 ratio. (For each reaction, prepare 20 µL: 2 µL of Proteinase K + 18 µL of PDB).
   2. Remove the tube from the magnetic stand after the final wash.
   3. Add 20 µL of freshly prepared Protein Digestion Working Solution to the bead pellet and gently mix.
   4. Place the PCR tube in a thermal cycler and incubate at 55 °C for 15 min.
   5. Place the PCR tube back on the magnetic stand. Wait until the solution clears (about 2 min).
   6. Carefully transfer the entire supernatant (about 20 µL) to a new 0.2 mL PCR tube.
      ​NOTE: RNA is now in the supernatant. Discard the beads.
6. RNA purification
   1. Add 20 µL of RNA Purification Solution (RPS) to each sample tube, negative control tube, and input tube.
   2. Add 160 µL of 100% ethanol.
   3. Vortex RNA Binding Beads to resuspend them.
   4. Add 2 µL of resuspended RNA Binding Beads to each tube.
   5. Mix thoroughly up and down at least 10 times.
   6. Incubate at room temperature for 5 min to allow RNA binding.
   7. Place the tube on the magnetic stand. Wait until the solution clears (about 2 min).
   8. Carefully discard the supernatant without disturbing the beads.
   9. Keep the tube on a magnetic stand and add 150 µL of freshly prepared 90% ethanol. Incubate for 30 s.
   10. Discard the 90% ethanol. Repeat the 90% ethanol wash one more time (two washes in total).
   11. Remove the tube from the magnetic stand.
   12. Add 13 µL of Elution Buffer to the dried beads.
   13. Incubate at room temperature for 5 min to elute RNA.
   14. Place the tube back on the magnetic stand. Wait until the solution clears (about 2 min).
   15. Carefully transfer the entire supernatant (about 13 µL) to a new RNase-free PCR tube.
   16. Use immediately or store RNA at -20 °C.

3. RT-qPCR

1. Reverse transcribe the m⁶A-enriched RNA into cDNA
   1. Use purified RNA from m⁶A-IP, Input, or IgG group for reverse transcription.
   2. Set up the reverse transcription (RT) reaction.
      NOTE: Ensure equal RNA input across all groups to allow reliable comparison in downstream qPCR analysis.
2. qPCR amplification
   1. Prepare qPCR reactions using SYBR Green chemistry. Add 10 µL of SYBR Green Master Mix (2×), 0.5 µL of forward primer (10 µM), 0.5 µL of reverse primer (10 µM), 7 µL of ddH₂O, and 2 µL of cDNA to each tube, mix thoroughly, and briefly centrifuge before loading into the qPCR instrument.
      NOTE: Set three replicate wells for each cDNA sample (Input, IgG, m⁶A-IP). Refer to Table 2 for the list of primers.
   2. Run the qPCR program.
      1. Perform the qPCR reactions on a real-time thermal cycler using the following program: set the initial denaturation at 95 °C for 30 s; run 40 cycles consisting of denaturation at 95 °C for 15 s, annealing at 58 °C for 10 s, and extension at 72 °C for 30 s.
      2. After amplification, conduct a melt curve analysis by setting denaturation at 95 °C for 15 s, cooling at 60 °C for 1 min, and then gradually increasing the temperature to 95 °C at a ramp rate of 0.5 °C/s with continuous fluorescence acquisition to confirm the specificity of the amplified products.
3. Data analysis
   1. Analyze Ct values of the target and control groups.
   2. Calculate enrichment using one of the following methods:
      1. Methods 1: % Input method
         %Input = 2^{[Ct(Input)−Ct(IP)]} × DF × 100%
         NOTE: Ct (Input) and Ct (IP) represent the cycle threshold values of the Input and IP samples, respectively, and dilution factor (DF) is the reciprocal of the input fraction used for qPCR (e.g., if 10% of total RNA was used as Input, then DF = 10).
      2. Methods 2: Comparative Ct method (2^^-ΔΔCt method)
         ΔCt_sample=Ct_IP−Ct_IgG
         Ct=ΔCt_treated−ΔCt_control, Fold=2^−ΔΔCt.
         NOTE: Keep RNA input amounts, reaction setup, and primer efficiency consistent across all samples.

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