RNA PolyA Tailing Assay to Qualitatively Analyze Circular RNA Manufacturing.

A covalently closed loop structure provides circular RNA (circRNA) with more stability than conventional RNAs in linear form, making circRNA an emerging tool in RNA therapeutics. The qualification and quantification of circRNA after production is critical for its design and effectiveness assessments, particularly when the following applications could be affected by byproduct RNAs. Despite PCR-based methods effectively detecting low-abundance circRNA, they are unsuitable for assessing uncircularized RNA in a mass production fraction to maintain quality control. Here, we present a straightforward protocol for evaluating uncircularized byproduct RNAs from circRNA production. This method enrolls the template-independent RNA polymerase activity to add adenine tails (polyA) to the 3' ends of a linear RNA, making it easy to distinguish trace byproducts or uncircularized RNA from a pool of mass circRNA products. With conventional linear RNA and RNase R-treated circRNA as the positive and negative controls, the purity of a circRNA preparation could be readily resolved. Regardless of circRNA production strategies, this protocol provides a reliable and practical way to ensure the consistent quality of homemade circRNAs or to recheck circRNA quality from commercial manufacturing. Key features • The RNA circularization assessment relies on detecting a free 3' end OH group of the non-circRNA in the circRNA products. • An RNA denaturing electrophoresis is required to detect nucleotide length changes of the non-circRNA post-3' end tailing. • Rather than quantitatively, the molecular weight changes qualitatively highlight the trace non-circRNA in a circRNA preparation. • While there are different techniques for producing circRNA, the tailing method is effective for most, detecting leftover linear RNA contaminants.