You can find our full CLIP protocol here adapted from Konig et al. Unlike RIP, CLIP provides information about the actual protein binding site on the RNA.ĭifferent types of CLIP exist, including high-throughput sequencing-CLIP (HITS-CLIP), photoactivatable-ribonucleoside enhanced CLIP (PAR-CLIP), and individual CLIP (iCLIP). This covalent bond is irreversible, allowing stringent purification conditions. For example, any novel results you obtain using RIP-seq should then be confirmed using RIP-qPCR.ĬLIP is an antibody-based technique used to study RNA-protein interactions related to RNA immunoprecipitation (RIP) but differs from RIP in that it uses UV radiation to cross-link RNA binding proteins to the RNA. Choose the best method based on the questions you want to answer and use multiple methods to confirm your results. The RNA isolated from your pull-down can be analyzed using several techniques. Knockdown cells are not recommended for negative control experiments.ĭownstream analysis. One or more negative controls should be maintained throughout the experiment, eg no antibody sample or immunoprecipitation from knockout cells or tissue. Use ultrapure distilled, DNase-free, RNase-free water to prepare buffers and solutions. Avoid contamination using RNase-free reagents such as RNase-free tips, tubes, and reagent bottles. Bottom method uses formaldehyde cross-linking. Top uses a native approach without cross-linking. Take a look at our full RIP protocol here.Īdapted from Khalila et al. RIP is one such protocol for the study of the physical association between individual proteins and RNA molecules. This new appreciation for the potential of RNA has lead to the development of novel methods allowing researchers to map RNA-protein interactions. For example, RNA-protein interactions can modulate mRNA and noncoding RNA function. Transcripts are detected by real-time PCR, microarrays or sequencing.īeyond transcription and subsequent translation, there is still is much more to the function of RNA.
The RNA binding protein (RBP) of interest is immunoprecipitated together with its associated RNA for identification of bound transcripts (mRNAs, non-coding RNAs or viral RNAs). RIP is an antibody-based technique used to map in vivo RNA-protein interactions. Many of our RNA modification antibodies are RabMAb ® monoclonal antibodies including key targets such as m6A, m1A, Mettl3, m2,2G, and many more. This gives you a consistent antibody and reproducible results throughout your project.
Recombinant RabMAb ® technology eliminates batch-to-batch variation by using the same immunogen for each round of antibody production. Once you have a working antibody you should ensure that between batches you are still getting specificity. It is important to be sure that the antibody you are using is specific for the modification you are interested in using the right controls and testing the antibody in your model system. RNA modification antibodies: technical considerationsĪntibody specificity and consistency. Click here for a full list of our RNA modification antibodies. This same m6A antibody also features in a Nature paper, looking at the role of m6A in mRNA stability (Mauer et al., 2017).
We have many well-cited and validated RNA modification antibodies available including our m6A antibody, cited in several great publications including a Nature Methods paper which uses it for single-base resolution sequencing of m6A (Linder et al., 2015). Getting antibodies that are specific to your RNA modification of choice can be difficult. In this section, we will go through some of these protocols and offer a few tips and advice for working with RNA modifications. The field of RNA modifications is relatively new but growing more and more every day. You can commonly find modifications in the anticodon loop of the tRNA, which help promote translation efficiency by aiding codon-anticodon interactions and preventing frameshifting (Stuart et al., 2003). The modifications on tRNA are incredibly diverse and require step-by-step formation by multiple enzymes 7. Of all the RNA species, tRNAs contain the most RNA modifications: almost one in five nucleotides within tRNAs are thought to contain RNA modifications (Kirchner et al., 2015). To find out more take a look at our RNA modifications poster The distribution of RNA modifications on mRNA and tRNAs.
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