The m6A modification is identified as the most prevalent and abundant modification in eukaryotic mRNAs and is found in multiple types of non-coding RNA. As interest in the dynamics and role of m6A has increased in recent years, we perform m6A RNA methylation analysis utilizing LC-MS/MS with MRM mode, for detecting and quantitating m6A.
Overview
More than 100 types of chemical modifications have been identified in RNA molecules. N6-Methyladenosine (m6A) was discovered and characterized in the 1970s, and is found in a number of organisms including viruses, yeast, insects, plants, and mammals. m6A is identified as the most conserved and abundant internal transcriptional modification, within eukaryotic mRNAs, microRNAs, and long non-coding RNAs (lncRNAs). RNA m6A is predominantly located in 3' UTR and near stop codons and translated near 5' UTR in a cap-independent manner, hence affecting RNA transcription, processing, translation and metabolism.
Mass spectrometry (MS) is a robust tool for qualitative and quantitative analysis of m6A modification and comparing m6A levels between different samples. We established a liquid chromatography-electrospray ionization-MS (LC-ESI-MS/MS) platform with MRM (Multiple Reaction Monitoring) mode, for the sensitive and accurate determination of RNA m6A. Our MS analysis can achieve both relative and absolute quantification of m6A modification. We provide sample-to-data m6A methylation analysis by MS. Additionally, we provide m6A-seq and MeRIP-seq for m6A RNA methylation mapping and quantification.
Features
Any Species
Transcriptome-Wide
Bioinformatics Analysis
Validated Process
This method can be applied to any species, from microorganisms to humans.
Profile transcriptome-wide m6A in mRNAs, miRNAs, lncRNAs, and other RNAs.
Data QC; detect and annotate RNA m6A; quantitate m6A; differential analysis.
Validated experimental process using LC-MS/MS with MRM.
Project Workflow
1. mRNA Isolation
Isolation of total RNA; enrichment of mRNA.
2. mRNA Digestion
Digest RNA with nuclease and alkaline phosphatase to generate single nucleosides.
3. LC-MS/MS
The nucleosides are subjected to LC-MS/MS analysis on QTRAP 6500 / 3200 with MRM mode.
4. Data Analysis
Visualize and preprocess results; relative and absolute quantification of m6A; differential analysis.
Sample Requirements
We work with a wide range of sample types including protein solution, fresh tissue, cultured cells, blood, and microbial sample. Please feel free to contact us for sample size.
Sample storage: extracted protein should be stored at -80°C. Avoid repeated freezing and thawing.
Shipping Method: When shipping the sample, it is stored in a 1.5 mL Eppendorf tube, sealed with a sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.
Deliverable: data QC report, MS results, integrated experimental report (materials, methodologies, and bioinformatics analysis).
References:
Yuan, Bi-Feng. "Liquid Chromatography-Mass Spectrometry for Analysis of RNA Adenosine Methylation." RNA Methylation. Humana Press, New York, NY, 2017. 33-42.2.
Chen X Y, Zhang J, Zhu J S. The role of m 6 A RNA methylation in human cancer. Molecular cancer, 2019, 18(1): 103.
* For Research Use Only. Not for use in diagnostic procedures.
