Circular RNA migration in agarose gel electrophoresis

Brian T Abe; R Alexander Wesselhoeft; Robert Chen; Daniel G Anderson; Howard Y Chang

doi:10.1016/j.molcel.2022.03.008

Circular RNA migration in agarose gel electrophoresis

Mol Cell. 2022 May 5;82(9):1768-1777.e3. doi: 10.1016/j.molcel.2022.03.008. Epub 2022 Mar 30.

Authors

Brian T Abe¹, R Alexander Wesselhoeft², Robert Chen³, Daniel G Anderson⁴, Howard Y Chang⁵

Affiliations

¹ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA 94305, USA; Division of Immunology and Rheumatology, Department of Medicine, Stanford University, Stanford, CA 94305, USA.
² Orna Therapeutics, Cambridge, MA 02139, USA.
³ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA 94305, USA.
⁴ Department of Chemical Engineering, Institute for Medical Engineering and Science, and David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
⁵ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA 94305, USA; Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA. Electronic address: howchang@stanford.edu.

Abstract

Circular RNAs are garnering increasing interest as potential regulatory RNAs and a format for gene expression. The characterization of circular RNA using analytical techniques commonly employed in the literature, such as gel electrophoresis, can, under differing conditions, yield different results when attempting to distinguish circular RNA from linear RNA of similar molecular weights. Here, we describe circular RNA migration in different conditions, analyzed by gel electrophoresis and high-performance liquid chromatography (HPLC). We characterize key parameters that affect the migration pattern of circular RNA in gel electrophoresis systems, which include gel type, electrophoresis time, sample buffer composition, and voltage. Finally, we demonstrate the utility of orthogonal analytical tests for circular RNA that take advantage of its covalently closed structure to further distinguish circular RNA from linear RNA following in vitro synthesis.

Keywords: E-gel; RNase H; RNase R; circRNA; circular RNA; electrophoresis.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Electrophoresis, Agar Gel / methods
Molecular Weight
RNA* / genetics
RNA, Circular* / genetics

Substances

RNA, Circular
RNA

Abstract

Publication types

MeSH terms

Substances

Grants and funding