Ulrix, L.; De Waele, J. H.; Vandesompele, J.; Hofmans, M.; De Moerloose, B.; Lammens, T.

Circular RNAs (circRNAs) are an emerging class of RNA molecules involved in development and disease. They are generated through back-splicing from pre-mRNA. RNaseR treatment, which degrades linear RNA while preserving circRNAs, is widely used for circRNA validation and enrichment. This short report demonstrated differential performance of two RNaseR enzymes from different suppliers, despite identical experimental conditions, indicating that these enzymes should not be considered interchangeable across suppliers. Importantly, these findings highlight that without demonstrating efficient linear RNA degradation, unchanged circRNA abundance after RNase R treatment is inherently uninformative and may contribute to false-positive circRNA validation or inadequate circRNA enrichment. We therefore highlight that RNaseR digestion should be consistently verified using a linear control, particularly when changing enzyme suppliers.