End-to-End Workflow for Viral RNA Modification Analysis

If the SARS-CoV-2 pandemic has taught us anything, it’s that research into RNA modifications is important now more than ever. Whether that implies studying the viral RNA itself to learn more about how different alterations and epigenetic changes make these viruses more resilient and infectious, or studying RNA gathered from cells and tissue to understand the effects these different pathogens have on host cells functions, the research endeavors are nearly endless. Often one of the more challenging parts of setting up an experiment is designing how each step of the process should be handled. In our effort to provide you with the complete solutions for epigenetic study, EpiGentek offers end-to-end workflow solutions in the form of kits and information. For your convenience, click the respective steps of the workflow above to jump to the section below and learn more about how each part plays into an experiment where RNA modifications of either cells, tissues or viruses can be studied.

The m6A RNA Cycle

The formation of m6A RNA is mediated by a multi-protein methyltransferase complex composed, in part, of METTL enzymes. The recent discoveries of these methylase “writers” and their associated demethylase “erasers” in mammals uncovered the reversibility of the m6A modification, as outlined in the following diagram. A methyl group is chemically added at the nitrogen-6 position of adenosine (A) residues by the METTL3/14 heterodimer to form N6-methyladenosine (m6A). Through FTO-mediated oxidative demethylation, m6A is converted in a step-wise manner to N6-hydroxymethyladenosine (hm6A) and subsequently N6-formyladenosine (f6A) before finally reverting back to A.

The Impact of m6A Modifications on the Life Cycles of RNA Viruses

Epigenetic modifications are known to influence the life cycles of RNA viruses like human coronavirus. Modified adenosines like N6-methyladenosine (m6A) are reported to affect the viability of specific RNA viruses by modulating viral cap structures, viral gene expression, virion progeny generation, innate sensing pathways, and the innate immune response. Gain or loss of m6A can result in significant functional changes to RNA viruses, altering host cell fusion/entry, replication, transmission, pathogen intensity, and immune evasion. The m6A epitranscriptome of host cells, which plays a role in host resistance, can also undergo alterations after viral infection. The SARS-CoV-2 genome, which spans over 29,800 nucleotides in length, contains more than 50 potential m6A sites based on the presence of specific sequence motifs for m6A modification by the RNA methylase complex METTL3/14, including GGACU(T), GGACA, and GGACC. Consequently, > 0.64% of all adenosines, or 0.18% of all bases, in SARS-CoV-2 RNA could be m6A. Quantification of m6A in total RNA and transcriptome-wide m6A mapping are thus essential for studying m6A-related viral function and corresponding mechanisms, as well as identifying novel targets for antiviral therapy.

RNA Extraction Kits

Whether you are starting with samples from either cells, tissues, or viruses, efficient RNA isolation is often the first step toward a successful experiment. This part of the workflow is crucial as adequate purity and yield are both essential requirements to ensure downstream applications run smoothly and accurately. That’s why it’s also important to note that the suggested kits and methods can change depending on your starting material:

For Viruses: when it comes to viral samples, on the other hand, challenges like low virus titers and high presence of contaminating host nucleic acids necessitate the use of supplementary techniques to ensure the yield and purity are in line with requirements downstream. In our recent bulletin article, we outline 10 ways in which this can be accomplished so you can work it into the method that works best for your study. One such way is by inoculating a cell culture for viral amplification, then harvesting the amplified virions for use with EpiGentek’s viral RNA extraction kits. NOTE: for viral RNA that is directly extracted from clinical specimens, additional processing is required to remove co-extracted host DNA and rRNA contaminants prior to m6A RNA enrichment.

For Cells / Tissue: processing not needed; thanks to the abundant amount of RNA in cells and tissue samples that can be acquired from extraction, there is less work required to result in obtaining suitable amounts of starting material for downstream applications. For cells or tissues, we suggest using our EpiQuik Total RNA Isolation Fast Kit or EpiQuik Magbeads Quick RNA Isolation Kit, both designed to work with such input samples.

Viral RNA Extraction Kits

Total RNA Extraction Kits

Nuclear Extraction Kit

The stage of nuclear extraction is important when dealing with cell or tissue starting materials that will eventually be analyzed for methylase or demethylase activity/inhibition. In this step, cells/tissues are treated with pre-lysis and lysis buffers to disrupt cellular and nuclear membranes, respectively, and extract the nuclear proteins for use downstream. Our proprietary kit below offers rapid 1-hour isolation that keeps enzymatic activity intact and includes all materials needed for a successful yield of nuclear protein extracts.

Global m6A/5mC RNA Quantification Kits

RNA methylation is a reversible post-translational modification that epigenetically impacts a great number of biological processes, and these can occur in both viral and non-viral-derived RNA. The most common of these is m6A methylation, which accounts for more than 80% of all RNA methylation, followed by 5-mC methylation, which can occur in various RNA molecules. If your study intends to look into the global methylation levels of m6A or 5-mC in RNA, our selection of colorimetric and fluorometric kits can help provide fast, accurate, global quantification. However, it must also be known that the process changes slightly if viral starting materials are used. If this is the case, you need to make sure the yield of total resulting RNA can be increased, as the low amount gathered from viral starting materials is often too low to use downstream.  We recommend referencing our recent bulletin article for various methods of virus purification for ideas on how to ensure the quality of total RNA to use later on in your workflow.

m6A Methylase & Demethylase Activity Assays

If you are planning on studying the RNA methylase or demethylase activity/inhibition, we recommend our robust nuclear extraction kits, which are fast and ensure that enzymatic activity stays intact after isolation.

Once the nuclear extracts are gathered, it is possible to move onto the assays that will help measure the activity/inhibition of the methylases or demethylases. Our colorimetric ELISA kits allow for high or manual throughput analysis of the extracted nuclear samples and are optimized to ensure background signals remain low, so measurements are accurate and reliable.

m6A Antibodies

As mentioned earlier, m6A is the most abundant mRNA modification and, as such, is typically a topic of interest for investigating researchers looking into epigenetic RNA modifications. Because of this, the mono- and polyclonal antibodies are often included in research studies of this kind to verify results and help understand how this particular modification affects the development of viruses and the effects of diseases once a host is infected. Our antibodies are validated in all applications listed and come in trial sizes for those labs looking to try out ideas before committing to purchase larger quantities.

MeRIP & m6A-seq Assays

Once other steps are completed in the workflow, a study might finally look to enrich for parts of a genome where m6A is present and prepare these segments for next generation sequencing by constructing a library. Our assay kits listed below both allow for enrichment of m6A RNA, while the EpiNext CUT&RUN RNA m6A-Seq kit gives the extra functionality for simultaneous library construction.

Studying COVID-19? See our new collection of SARS-CoV-2 Related Antibodies, Proteins & Assays.

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