Validation of Circular RNAs Using RT?qPCR After Effective Removal of Linear RNAs by Ribonuclease R

Circular RNAs (circRNAs) are a class of endogenous noncoding that have been shown to play role in normal development, homeostasis, and disease, including cancer. CircRNAs formed through process called back-splicing, which results covalently closed loop with nonlinear back-spliced junction (BSJ). In general, circRNA BSJs predicted RNA sequencing data using one numerous detection algorithms. Selected circRNAs then typically validated an orthogonal method such as reverse transcription quantitative PCR (RT-qPCR) circRNA-specific primers. However, linear transcripts originating from trans-splicing can lead false-positive signals both RT-qPCR experiments. Therefore, it is essential perform the validation step only after degraded exonuclease ribonuclease R (RNase R). Several RNase protocols available for or RT-qPCR. These protocols—which vary enzyme concentration, input amount, incubation times, cleanup steps—typically lack detailed standard protocol fail provide range conditions deliver accurate results. As such, some use concentrations too high, resulting partial degradation target circRNAs. Here, we describe optimized workflow validation, combining treatment First, outline steps primer design qPCR assay validation. Then, total and, importantly, subsequent buffer step. Lastly, discuss downstream analyses. © 2021 Wiley Periodicals LLC. Basic Protocol 1: CircRNA 2: treatment, cleanup,