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Practical guide for circular RNA analysis: Steps, tips, and resources

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Abstract Recent technological advances in RNA sequencing and analysis have allowed an increasingly thorough investigation of a previously unexplored class of transcripts, circular (circ)RNAs. Accumulating evidence suggests that circRNAs have unique functions which often rely on their association with microRNAs and RNA‐binding proteins. Through these interactions, circRNAs have been implicated in major cellular processes and hence in the pathophysiology of a range of diseases. Here, we provide guidelines to consider when developing studies on circRNAs, including detecting and selecting the circRNAs, identifying their binding partners and sites of interaction, modulating circRNA levels, assessing copy numbers and stoichiometry, and addressing other points unique to circRNA analysis. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs
Systematic circular RNA (circRNA) analysis, from identification to function. First, a circRNA is identified depending on the biological question at hand. Experimentally, this can be achieved by enriching circRNAs from a pool of RNAs and using a high‐throughput method to identify them (e.g., RNA‐seq or microarray) (step 1). After validation (step 2), the functional influence of the circRNA can be assessed by silencing and overexpressing it in an appropriate model system (step 3). By employing affinity pulldown assays, the molecules interacting with the circRNA—proteins and microRNAs, for example—can be identified (step 4), and the stoichiometry of the circRNA and the interacting molecules can be assessed (step 5). It is then important to identify and ablate the sites of interaction of the circRNA with associated molecules (step 6). Finally, rescue experiments that interfere with the interaction of the circRNA with associated factors offer valuable mechanistic insight into the function of the circRNA (step 7). ASO, antisense oligonucleotide; circFISH, circular RNA fluorescence in situ hybridization; ddPCR, Droplet Digital PCR; RIP, RNP immunoprecipitation; RPAD, RNase R treatment followed by polyadenylation and poly(A) + RNA depletion; RT‐qPCR, reverse transcription followed by quantitative PCR analysis
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Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs

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