Name: MeRIP-qPCR Assay for Detecting m6A Modification Levels of Specific RNA in Osteosarcoma Cells (Video) | JoVE Journal
Uploaded: 2025-12-30T13:55:00.000+00:00
Duration: 5 min 16 s
Description: Learn MeRIP-qPCR to detect m6A modification levels in specific RNA transcripts using osteosarcoma cells. This sensitive method offers a low-cost alternative to sequencing-based approaches for cancer research.

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5 minutes

This study was supported by grants from National Nature Science Foundation (82174408, 82374477, 82474535 and 82205145).

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 &#176;C before starting.
<ol>
	<li>Prepare the cells.
	<ol>
		<li>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.</li>
		<li>Transfer the cell suspension into a 1.5 mL microcentrifuge tube.</li>
		<li>Centrifuge at 1000&#160;&#215;&#160;g for 5 min at 4 &#176;C. 
		NOTE: Ensure that the rotor is balanced and the lid is closed securely.</li>
		<li>Carefully take out the microcentrifuge tube after centrifugation.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Lyse cells.
	<ol>
		<li>Add 300 &#181;L of Lysis Buffer to the cell pellet.</li>
		<li>Mix thoroughly by inverting the tube 30 times until the solution appears clear and viscous with no visible clumps.</li>
		<li>Incubate the tube at room temperature for 2 min.</li>
	</ol>
	</li>
	<li>Precipitate RNA.
	<ol>
		<li>Add 300 &#181;L of 100% ethanol to the lysate (equal volume of Lysis Buffer).</li>
		<li>Gently pipette up and down immediately (about 8-10 times) to fully mix.</li>
	</ol>
	</li>
	<li>Perform RNA column binding.
	<ol>
		<li>Place an RNA purification column into a provided 2 mL collection tube.</li>
		<li>Transfer the entire mixture to the purification column. 
		NOTE: Avoid touching the white membrane in the center.</li>
		<li>Close the cap and centrifuge at 12,000 &#215;&#160;g for 1 min.</li>
		<li>Discard the lower liquid and gently tap the collection tube upside down on absorbent paper to remove residual liquid.</li>
		<li>Return the purification column to the collection tube.</li>
	</ol>
	</li>
	<li>Wash impurities.
	<ol>
		<li>Add 500 &#181;L of Wash Buffer ( Nuclease-free water) to the purification column. 
		NOTE: Avoid touching the white membrane in the center.</li>
		<li>Centrifuge at 12,000 &#215;&#160;g for 1 min and discard the lower liquid.</li>
		<li>Centrifuge again at 12,000 &#215;&#160;g for 1 min to remove residual liquid.</li>
		<li>Transfer the purification column into a clean 1.5 mL microcentrifuge tube.</li>
		<li>Open the cap and let the column stand for 2 min at room temperature.</li>
	</ol>
	</li>
	<li>Elute RNA.
	<ol>
		<li>Place the purification column into a new 1.5 mL microcentrifuge tube.</li>
		<li>Add 25 &#181;L of Elution Buffer directly onto the center of the membrane.</li>
		<li>Incubate at room temperature for 5 min.</li>
		<li>Centrifuge at 12,000&#160;&#215;&#160;g for 1 min to collect RNA in the lower tube.</li>
		<li>Reapply the eluate to the membrane and centrifuge again at 12,000&#160;&#215;&#160;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.</li>
	</ol>
	</li>
	<li>Measure RNA concentration.
	<ol>
		<li>Rinse the spectrophotometer sample port with 1.5 &#181;L of Elution Buffer.</li>
		<li>Use 1.5 &#181;L of Elution Buffer as a blank. Confirm that the reading is close to zero.</li>
		<li>Load 1.5 &#181;L of RNA sample and measure the concentration.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Methylated RNA immunoprecipitation (MeRIP)
<ol>
	<li>Preparation of immunocapture mixture
	<ol>
		<li>Obtain a new 0.2 mL PCR tube.</li>
		<li>Add components as listed in Table 1 (example for one reaction).</li>
	</ol>
	</li>
	<li>Immunocapture
	<ol>
		<li>Close the tube cap tightly. Place the PCR tube on a rotator or rolling shaker.</li>
		<li>Incubate at room temperature for 90 min. 
		NOTE: Ensure that the solution is mixed continuously and gently.</li>
	</ol>
	</li>
	<li>RNA cleavage
	<ol>
		<li>After incubation, add 10 &#181;L of Nuclear Digestion Enhancer (NDE) and 2 &#181;L of Cleavage Enzyme Mix (CEM) to each tube.</li>
		<li>Mix gently by pipetting up and down 5 to 6 times.</li>
		<li>Incubate at room temperature for 4 min.</li>
		<li>Preparation of the Input RNA control
		<ol>
			<li>To ensure consistency between immunoprecipitation (IP) and Input samples, process a portion of RNA in parallel as the Input control.</li>
			<li>Transfer 1 &#181;g of total RNA (10% input) from each sample into a separate PCR tube. Add 20 &#181;L of immuno capture buffer (ICB), 1.5 &#181;L of NDE, 1 &#181;L of CEM, and incubate at room temperature for 2 min.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Magnetic enrichment and washing
	<ol>
		<li>Place the PCR tube on a magnetic stand.</li>
		<li>Allow the tube to sit for 2 min until the solution becomes clear and the beads are captured on the tube wall.</li>
		<li>Carefully aspirate and discard the supernatant without disturbing the beads.</li>
		<li>Keep the tube on a magnetic stand and add 150 &#181;L of Wash Buffer (WB).</li>
		<li>Remove the tube from the magnetic stand and tilt it upside down gently to fully resuspend the beads.</li>
		<li>Return the tube to the magnetic stand. Wait 1-2 min until the solution clears, then discard the supernatant.</li>
		<li>Repeat the wash step three times.</li>
		<li>Wash the beads once more with 150 &#181;L of Protein Digestion Buffer (PDB) using the same method.</li>
	</ol>
	</li>
	<li>Release and recovery of enriched RNA
	<ol>
		<li>Prepare Protein Digestion Working Solution by mixing Proteinase K and PDB in a 1:9 ratio. (For each reaction, prepare 20 &#181;L: 2 &#181;L of Proteinase K + 18 &#181;L of PDB).</li>
		<li>Remove the tube from the magnetic stand after the final wash.</li>
		<li>Add 20 &#181;L of freshly prepared Protein Digestion Working Solution to the bead pellet and gently mix.</li>
		<li>Place the PCR tube in a thermal cycler and incubate at 55 &#176;C for 15 min.</li>
		<li>Place the PCR tube back on the magnetic stand. Wait until the solution clears (about 2 min).</li>
		<li>Carefully transfer the entire supernatant (about 20 &#181;L) to a new 0.2 mL PCR tube. 
		&#8203;NOTE: RNA is now in the supernatant. Discard the beads.</li>
	</ol>
	</li>
	<li>RNA purification
	<ol>
		<li>Add 20 &#181;L of RNA Purification Solution (RPS) to each sample tube, negative control tube, and input tube.</li>
		<li>Add 160 &#181;L of 100% ethanol.</li>
		<li>Vortex RNA Binding Beads to resuspend them.</li>
		<li>Add 2 &#181;L of resuspended RNA Binding Beads to each tube.</li>
		<li>Mix thoroughly up and down at least 10 times.</li>
		<li>Incubate at room temperature for 5 min to allow RNA binding.</li>
		<li>Place the tube on the magnetic stand. Wait until the solution clears (about 2 min).</li>
		<li>Carefully discard the supernatant without disturbing the beads.</li>
		<li>Keep the tube on a magnetic stand and add 150 &#181;L of freshly prepared 90% ethanol. Incubate for 30 s.</li>
		<li>Discard the 90% ethanol. Repeat the 90% ethanol wash one more time (two washes in total).</li>
		<li>Remove the tube from the magnetic stand.</li>
		<li>Add 13 &#181;L of Elution Buffer to the dried beads.</li>
		<li>Incubate at room temperature for 5 min to elute RNA.</li>
		<li>Place the tube back on the magnetic stand. Wait until the solution clears (about 2 min).</li>
		<li>Carefully transfer the entire supernatant (about 13 &#181;L) to a new RNase-free PCR tube.</li>
		<li>Use immediately or store RNA at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
3. RT-qPCR
<ol>
	<li>Reverse transcribe the m6A-enriched RNA into cDNA
	<ol>
		<li>Use purified RNA from m6A-IP, Input, or IgG group for reverse transcription.</li>
		<li>Set up the reverse transcription (RT) reaction. 
		NOTE: Ensure equal RNA input across all groups to allow reliable comparison in downstream qPCR analysis.</li>
	</ol>
	</li>
	<li>qPCR amplification
	<ol>
		<li>Prepare qPCR reactions using SYBR Green chemistry. Add 10 &#181;L of SYBR Green Master Mix (2&#215;), 0.5 &#181;L of forward primer (10 &#181;M), 0.5 &#181;L of reverse primer (10 &#181;M), 7 &#181;L of ddH2O, and 2 &#181;L 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.</li>
		<li>Run the qPCR program.
		<ol>
			<li>Perform the qPCR reactions on a real-time thermal cycler using the following program: set the initial denaturation at 95 &#176;C for 30 s; run 40 cycles consisting of denaturation at 95 &#176;C for 15 s, annealing at 58 &#176;C for 10 s, and extension at 72 &#176;C for 30 s.</li>
			<li>After amplification, conduct a melt curve analysis by setting denaturation at 95 &#176;C for 15 s, cooling at 60 &#176;C for 1 min, and then gradually increasing the temperature to 95 &#176;C at a ramp rate of 0.5 &#176;C/s with continuous fluorescence acquisition to confirm the specificity of the amplified products.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Analyze Ct values of the target and control groups.</li>
		<li>Calculate enrichment using one of the following methods:
		<ol>
			<li>Methods 1: % Input method 
			%Input = 2[Ct(Input)&#8722;Ct(IP)] &#215; DF &#215; 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).</li>
			<li>Methods 2: Comparative Ct method (2^-&#916;&#916;Ct method) 
			&#916;Ctsample=CtIP&#8722;CtIgG 
			Ct=&#916;Cttreated&#8722;&#916;Ctcontrol, Fold=2&#8722;&#916;&#916;Ct. 
			NOTE: Keep RNA input amounts, reaction setup, and primer efficiency consistent across all samples.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol><li>Ma, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+interplay+between+m6A+RNA+methylation+and+noncoding+RNA+in+cancer%5BTitle%5D)+AND+%22J+Hematol+Oncol%22%5BJournal%5D)">The interplay between m6A RNA methylation and noncoding RNA in cancer.</a> J Hematol Oncol. 12 (1), 121(2019).</li>
<li>Zhu, Z. M., Huo, F. C., Zhang, J., Shan, H. J., Pei, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crosstalk+between+m6A+modification+and+alternative+splicing+during+cancer+progression%5BTitle%5D)+AND+%22Clin+Transl+Med%22%5BJournal%5D)">Crosstalk between m6A modification and alternative splicing during cancer progression.</a> Clin Transl Med. 13 (10), e1460(2023).</li>
<li>Hu, T., Wang, G., Wang, D., Deng, Y., Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((m6A+methylation+modification%3A+Potential+pathways+to+suppress+osteosarcoma+metastasis%5BTitle%5D)+AND+%22Int+Immunopharmacol%22%5BJournal%5D)">m6A methylation modification: Potential pathways to suppress osteosarcoma metastasis.</a> Int Immunopharmacol. 145, 113759(2025).</li>
<li>Nagarajan, A., Janostiak, R., Wajapeyee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dot+blot+analysis+for+measuring+global+N6-methyladenosine+modification+of+RNA%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Dot blot analysis for measuring global N6-methyladenosine modification of RNA.</a> Methods Mol Biol. 1870, 263-271 (2019).</li>
<li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+progress+in+strategies+to+profile+transcriptomic+m6A+modifications%5BTitle%5D)+AND+%22Front+Cell+Dev+Biol%22%5BJournal%5D)">Current progress in strategies to profile transcriptomic m6A modifications.</a> Front Cell Dev Biol. 12, 1392159(2024).</li>
<li>McIntyre, A. B. R., Gokhale, N. S., Cerchietti, L., Jaffrey, S. R., Horner, S. M., Mason, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Limits+in+the+detection+of+m6A+changes+using+MeRIP%2Fm6A-seq%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Limits in the detection of m6A changes using MeRIP/m6A-seq.</a> Sci Rep. 10 (1), 6590(2020).</li>
<li>Meyer, K. D., Saletore, Y., Zumbo, P., Elemento, O., Mason, C. E., Jaffrey, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+analysis+of+mRNA+methylation+reveals+enrichment+in+3%27+UTRs+and+near+stop+codons%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons.</a> Cell. 149 (7), 1635-1646 (2012).</li>
<li>Zheng, H. X., Zhang, X. S., Sui, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+the+profiling+of+N6-methyladenosine+%28m6A%29+modifications%5BTitle%5D)+AND+%22Biotechnol+Adv%22%5BJournal%5D)">Advances in the profiling of N6-methyladenosine (m6A) modifications.</a> Biotechnol Adv. 45, 107656(2020).</li>
<li>Matsuzawa, S., Wakata, Y., Ebi, F., Isobe, M., Kurosawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+validation+of+monoclonal+antibodies+against+N6-methyladenosine+for+the+detection+of+RNA+modifications%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Development and validation of monoclonal antibodies against N6-methyladenosine for the detection of RNA modifications.</a> PLoS One. 14 (10), e0223197(2019).</li>
<li>Law, J., Günther, S., Watanabe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epitranscriptome+mapping+of+N6-methyladenosine+using+m6A+immunoprecipitation+with+high-throughput+sequencing+in+skeletal+muscle+stem+cells%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Epitranscriptome mapping of N6-methyladenosine using m6A immunoprecipitation with high-throughput sequencing in skeletal muscle stem cells.</a> Methods Mol Biol. 2640, 431-443 (2023).</li>
<li>Mathur, L., Jung, S., Jang, C., Lee, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+analysis+of+m6A+RNA+modification+by+LC-MS%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Quantitative analysis of m6A RNA modification by LC-MS.</a> STAR Protoc. 2 (3), 100724(2021).</li>
<li>Slama, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+enrichment+factors+for+modified+RNA+in+MeRIP+experiments%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Determination of enrichment factors for modified RNA in MeRIP experiments.</a> Methods. 156, 102-109 (2019).</li>
<li>Guo, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-molecule+m6A+detection+empowered+by+endogenous+labeling+unveils+complexities+across+RNA+isoforms%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">Single-molecule m6A detection empowered by endogenous labeling unveils complexities across RNA isoforms.</a> Mol Cell. 85 (6), 1233-1246.e7 (2025).</li>
</ol>

The authors have nothing to disclose.

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...

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.

<table><tbody><tr><td>100% ethanol</td><td>Shanghai Titan Scientific Co.,Ltd., America</td><td>1158566</td><td></td></tr><tr><td>143B</td><td>ATCC, America</td><td>ATCC CRL-8303TM</td><td></td></tr><tr><td>2&amp;times;SYBR Green qPCR Mix</td><td>Beijing Qihengxing Biotechnology Co., Ltd., China</td><td>FS-Q1002</td><td></td></tr><tr><td>Denovix DS-11&amp;nbsp;</td><td>Denovix</td><td>DS-11&amp;nbsp;</td><td>Spectrophotometer</td></tr><tr><td>DMEM medium</td><td>WISENT CORPORATION, China</td><td>319-005-CL</td><td></td></tr><tr><td>DMEM/F-12 medium</td><td>Gibco, America</td><td>11320033</td><td></td></tr><tr><td>EpiQuik CUT&amp;amp;RUN m6A RNA Enrichment (MeRIP) Kit</td><td>Epigentek, America</td><td>P-9018-24</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>WISENT CORPORATION, China</td><td>086-150</td><td></td></tr><tr><td>hFOB1.19</td><td>Cell Bank of the Chinese Academy of Sciences, China</td><td>GNHu14</td><td></td></tr><tr><td>m&lt;sup&gt;6&lt;/sup&gt;A RNA Methylation Assay Kit</td><td>Abcam, England</td><td>ab185912</td><td></td></tr><tr><td>microRNA Reverse Transcription Kit</td><td>EZBioscience-EZB, America</td><td>A0010CGQ</td><td></td></tr><tr><td>Real-Time System</td><td>BIO-RAD, America</td><td>CFX96</td><td></td></tr><tr><td>Total RNA Extraction Kit</td><td>EZBioscience-EZB, America</td><td>B0004DP</td><td></td></tr><tr><td>TrypLE Express Enzyme</td><td>Gibco, America</td><td>12604021</td><td></td></tr></tbody></table>

merip-qpcr assay for detecting m6a modification levels of specific rna in osteosarcoma cells

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

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.

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....

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

RNA epitranscriptomic modification plays a critical role in the initiation and development of various cancers, among which N6-methyladenosine (m6A) is the ...

My research focuses on osteosarcoma. I'm trying to address the advantages of MeRIP-qPCR compared with other technical and future research directions. To begin, remove the culture medium from a 3.5 centimeter dish containing osteosarcoma cells.Gently wash once with pre-chilled PBS. Add an appropriate volume of RNA lysis buffer directly to the dish to fully lyse the cells. Gently pipette the suspension to homogenize.Transfer the cell suspension into a 1.5 milliliter microcentrifuge tube. Then centrifuge the microcentrifuge tube at 1, 000 G for five minutes at four degrees Celsius. Carefully remove the microcentrifuge tube after centrifugation.Using a one milliliter pipette, aspirate the supernatant completely without disturbing the pellet at the bottom. Then add 300 microliters of lysis buffer to the cell pellet. Mix thoroughly by inverting the tube 30 times until the solution appears clear and viscous with no visible clumps.Then incubate the tube at room temperature for two minutes and proceed with RNA precipitation and purification. For methylated RNA immunoprecipitation, add the listed reagents to the tube. Close the cap of the PCR tube tightly for immuno capture.Then place the PCR tube on a rotator or rolling shaker and incubate. Now transfer the PCR tube onto a magnetic stand. Allow the tube to sit for two minutes until the solution becomes clear and the beads are captured on the tube wall.Then carefully aspirate and discard the supernatant without disturbing the beads. While keeping the tube on the magnetic stand, add 150 microliters of wash buffer. Remove the tube from the magnetic stand and tilt it upside down gently to fully resuspend the beads.Return the tube to the magnetic stand. After two minutes when the solution clears, discard the supernatant. Now add 20 microliters of freshly prepared protein digestion working solution to the beads and incubate the PCR tube in a thermal cycler.After incubation, place the tube back on the magnetic stand and carefully transfer the entire supernatant to a new 0.2 milliliter PCR tube. Add 20 microliters of RNA purification solution to each sample tube, negative control tube, and input tube. Then pipette 160 microliters of 100%ethanol.Vortex the RNA binding beads to resuspend them. Then transfer two microliters of resuspended RNA binding beads to each tube. Place the tube on a magnetic stand, discard the supernatant and add 150 microliters of freshly prepared 90%ethanol.After discarding the ethanol, pipette 13 microliters of elution buffer to the dried beads and incubate at room temperature for five minutes. Then carefully transfer the entire supernatant to a new RNAse-free PCR tube for further analysis, MeRIP-qPCR was used to assess N6-methyladenosine modification levels in the c-Myc RNA transcript in osteosarcoma cells. c-Myc enrichment using the anti-N6-methyladenosine antibody was significantly higher than with immunoglobulin G control in both hFOB1.19 and 143B cells.The c-Myc N6-methyladenosine modification level was significantly higher in 143B cells than in hFOB1.19 cells. MeRIP-qPCR sensitively detects low abundance m6A enables regional localization and offers a low cost alternative to sequencing-based approaches with antibody enrichment efficiency. Future studies are expected to further integrate MeRIP-qPCR with nano-sequencing to improve accuracy, resolution, and the foundational interpretation of m6A modifications.

merip-qpcr-assay-for-detecting-m6a-modification-levels-specific-rna

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We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

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

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The present protocol describes a method of RNA fluorescence in situ hybridization to localize the lncRNAs in human osteosarcoma cells.

RNA Fluorescence In Situ Hybridization for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

The protocol describes a tandem immunofluorescence hybridization assay with formalin-fixed paraffin-embedded tissue to detect and quantify microRNA and multiplexed proteins simultaneously.

MicroRNA Amplification and Recognition through Locked-nucleic-acid In situ Hybridization as a Novel Detection and Quantification Method

Here, we present a protocol to semi-quantitatively assess global m⁶A levels using dot blot. Total RNA is extracted, denatured, spotted on a nylon membrane, probed with anti-m⁶A antibody, and visualized by chemiluminescence. Signal intensity, quantified by ImageJ grayscale analysis, reflects relative methylation abundance, providing a reproducible workflow for research.

Dot Blot Assay for Detecting Global N6-Methyladenosine RNA Modification Levels

This experimental protocol provides a stable, cost-effective, and efficient method that combines lncRNA knockdown with colony-forming assays to quantify the effect of lncRNA on osteosarcoma cell proliferation.

Colony Formation Assay Detecting the Proliferative Capacity of LncRNA-knockdown Osteosarcoma Cells

Learn MeRIP-qPCR to detect m6A modification levels in specific RNA transcripts using osteosarcoma cells. This sensitive method offers a low-cost alternative to sequencing-based approaches for cancer research.

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

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