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

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