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