Complete Solutions for RNA Methylation Analysis

Expand upon your understanding of RNA with EpiGentek’s complete suite of RNA methylation products

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What is RNA Methylation?

RNA methylation is a type of posttranscriptional modification made to RNA that affects gene regulation without altering the structural genetic sequence. It is recognized in numerous species of organism, including viruses, and strongly associates with important biological processes. Like DNA methylation, RNA methylation is reversible, abundant in life, and primarily involves the addition of methyl groups. However, its distribution in different types of RNA is not uniform. RNA methylation occurs in tRNA, rRNA, mRNA, tmRNA, snRNA, snoRNA, miRNA, and viral RNA. Two of the more familiar modifications of mRNA include N6-methyladenosine (m6A) and 5-methylcytosine (5mC).

Of all the chemically distinct RNA modifications, N6-methyladenosine (m6A) is the most common. It involves methylation of the adenosine base at the nitrogen-6 position. Often called the “fifth RNA base”, m6A accounts for over 80% of all RNA methylation present in eukaryotes. In particular, this RNA modification is thought to be important for mRNA regulation due to enrichment of m6A at 3’UTRs. It has also been associated with various types of cancers, including leukemia, breast cancer, and prostate cancer.

Another abundant epigenetic mark found in various RNA molecules is 5-methylcytosine (5mC), which involves adding a methyl group at the 5-carbon ring. It is reported in several different RNA species, including rRNAs, tRNAs, eRNAs, as well as mRNAs, and various non-coding RNAs. The ideal way to detect the 5mC modification in RNA at a single point is through bisulfite conversion followed by high-throughput sequencing.

Which RNA Methylation Tool is Right for Your Experiment?

The importance of RNA modifications, in particular RNA methylation, is evident. However, the field of studying RNA methylation is only in its infancy. Many established detection methods rely heavily on antibody-dependent transcriptome-wide techniques, most of which focus on the occurrence of m6A in RNA due to its well-known biological role. 5mC RNA detection techniques, including bisulfite sequencing, also attract interest as 5mC RNA has been linked to multiple cellular processes and tumorigenesis. Newer biochemical approaches, combined with high‐throughput sequencing, are making it possible to study whole transcriptomes as well as map individual modifications, revealing the true function of RNA‐modifying enzymes or protein binding factors.

Before you begin your RNA methylation research project, we suggest reviewing any available literature related to your proposed experiment. If other researchers have done similar work, you should take advantage of their information and use it as a base for your study, making sure to review it carefully for any potential obstacles.

If you are looking to analyze the global levels of m6A or 5mC modification in RNA, we suggest starting with a simple pre-screen of methylation status in your samples. These methods are available with our popular m6A RNA methylation quantification kit and 5-mC RNA methylation ELISA kit. For directly targeting either m6A or 5mC, we suggest using our m6A enrichment kits or bisulfite conversion kits.

See our RNA methylation resource center for more helpful information on RNA Methylation. To get an idea of our offerings, we also break down the benefits of our RNA Methylation products for your convenience. For studies involving viral and host RNA modifications, visit our epigenetic viral RNA modification workflow page.

RNA Methylation Studies Step Details

RNA Isolation

Isolating RNA is a critical first step in any RNA methylation study. Our RNA preparation kits allow for the quick and efficient isolation of total RNA directly from samples such as tissue, cultured cells, and cell-free liquid specimens (specifically saliva and nasopharyngeal swabs). The purified RNA can be used for various routine applications, such as RT-PCR, cDNA synthesis, Northern blotting, differential display, primer extension, and mRNA selection.

Global m6A & 5mC RNA Quantification

EpiGentek's m6A and 5mC RNA methylation kits make it easy for researchers to determine how RNA methylation influences their research project. Our kits allow for the direct quantification of either N6-Methyladenosine (m6A) or measuring global 5-mC RNA using total RNA isolated from any species, including mammals, plants, fungi, bacteria, and viruses.

RNA Bisulfite Conversion & cDNA Synthesis

Bisulfite treatment techniques, also known as bisulfite conversion, are the most ideal approaches for preparing RNA for site-specific methylation analysis. This process converts cytosine into uracil, leaving methylated cytosine intact which is then easily distinguishable via high-throughput sequencing. Bisulfite converted RNA can also be used to generate cDNA for use in various downstream applications to analyze methylation condition.

Library Preparation for NGS

Library preparation is a critical step for next generation sequencing (NGS). We offer many inexpensive solutions for NGS, including kits that conveniently construct high-quality RNA/ DNA libraries from difficult samples that can be used in various Illumina-based platforms.

MeRIP & m6A-Seq Assays

Get high resolution mapping of m6A RNA using our CUT&RUN RNA m6A enrichment kits. With a unique nucleic acid cleavage enzyme mix, these kits isolate only RNA segments at the antibody binding locations and simultaneously cleave/remove the segments of RNA sequence just under the target area. Both our kits can enrich RNA fragments containing the m6A modification from low input RNA, with minimal non-specific background levels. The CUT&RUN RNA m6A-Seq Kit includes the library preparation step as an added benefit.

Antibodies to Study RNA Methylation

N6-methyladenosine (m6A) is one of the most common and abundant modifications found in RNA molecules present in eukaryotes. The chemical properties of m6A make it challenging to distinguish; therefore, methods to analyze this modification are highly dependent upon the quality of the ant-m6A antibody used. Both our polyclonal and monoclonal m6A antibodies can be reliably used for the specific detection of m6A in RNA. They have been thoroughly tested and are guaranteed to work in their respective applications.