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.
Orna Therapeutics, Cambridge, MA 02139, USA.
Mol Cell. 2022 May 5;82(9):1768-1777.e3. doi: 10.1016/j.molcel.2022.03.008. Epub 2022 Mar 30.
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.
环状 RNA 作为潜在的调控 RNA 和基因表达的一种形式，正受到越来越多的关注。使用文献中常用的分析技术（如凝胶电泳）来表征环状 RNA，在尝试区分分子量相似的环状 RNA 和线性 RNA 时，在不同条件下可能会产生不同的结果。在这里，我们通过凝胶电泳和高效液相色谱（HPLC）描述了不同条件下环状 RNA 的迁移。我们描述了影响凝胶电泳系统中环状 RNA 迁移模式的关键参数，包括凝胶类型、电泳时间、样品缓冲液组成和电压。最后，我们展示了利用环状 RNA 共价封闭结构的正交分析测试的实用性，该测试可进一步区分体外合成的环状 RNA 和线性 RNA。
