RNA methylation is a reversible chemical modification involved in the epigenetic regulation of numerous biological processes. It occurs in different RNA types (tRNA, rRNA, mRNA, tmRNA, snRNA, snoRNA, miRNA, viral RNA), and different catalytic strategies are employed for modifying RNA by a variety of methyltransferases.
Researchers have an assortment of tools and techniques at their disposal to analyze RNA methylation at genome-wide, gene-specific, and single-base resolution levels. These methods generally rely on the use of antibodies (e.g., ELISA, MeRIP-seq, miCLIP) that target particular modifications, although there are a number of antibody-free options (e.g., bisulfite conversion, SCARLET, DART-seq) available. Whichever experimental route investigators decide to pursue in their epitranscriptomic studies, there are certain precautions that should be taken to ensure reliable and consistent results.
Stability issues
The hydroxyl group at the 2' position of the ribonucleotide sugar moiety renders RNA susceptible to both spontaneous and enzyme-mediated hydrolytic cleavage. Thus, RNA is chemically unstable in comparison to DNA and therefore more demanding to work with. Tissue samples may be stabilized with liquid nitrogen or with a commercial stabilization compound in order to protect RNA prior to extraction. RNA should ideally be extracted from freshly harvested specimens and in a timely manner. If not immediately used, extracts should be stored in single-use aliquots at low temperature (-80°C).
Purity and quality
Antibody-based methods for detecting methylation cannot distinguish between modified DNA and RNA. As such, input RNA should be highly pure (e.g., A260/280 ratio = 1.9 - 2.1). The incorporation of DNase treatment into RNA extraction protocols can help minimize DNA contamination. For high-quality total RNA preps, electrophoresis analysis should reveal prominent 18S and 28S rRNA bands, with a 28S/18S band intensity ratio greater than 2.
Immunoassay considerations
Like DNA, RNA contains a diversity of methylation types (e.g., 5mC, m6A). For immunoassay-based detection, it is hence essential to carefully pick a primary antibody that is highly specific for the target methylation and displays no cross-reactivity with off-target modifications. Additionally, the antibody of choice should be validated for use in the intended application (e.g. ELISA, immunoprecipitation).
Ribonuclease dilemmas
The prevalence of active RNases within samples and in the surrounding environment, especially on hands and surfaces, poses an issue for RNA integrity. In addition to maintaining proper aseptic technique, it should be ensured that RNase-free reagents are strictly used, including water, buffers, pipette tips, glassware, and instruments, and workspaces should be appropriately treated to eliminate RNases.
As pioneers in the R&D of epigenetics-based research products, EpiGentek has leveraged its proprietary and innovative technologies to develop convenient and dependable assays with the fastest protocols for global methylated RNA and methyltransferase activity assessment, MeRIP, and bisulfite conversion/NGS library construction, providing complete solutions for your RNA methylation studies.
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