1. A SYBR Gold-based Label-free in vitro Dicing Assay
- Author
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Qian Wang, Yan Xue, Laixing Zhang, Zhenhui Zhong, Suhua Feng, Changshi Wang, Lifan Xiao, Zhenlin Yang, C. Harris, Zhe Wu, Jixian Zhai, Maojun Yang, Sisi Li, Steven Jacobsen, and Jiamu Du
- Subjects
Biology (General) ,QH301-705.5 - Abstract
In Arabidopsis, DICER-LIKE PROTEIN 3 (DCL3) cuts the substrate pre-siRNA into a product siRNA duplex, encompassing one 23-nt strand and one 24-nt strand. To monitor the separation of the siRNA duplex with only 1-nt difference, we developed this protocol to evaluate the in vitro dicing activity of DCL3. The method can be applied for measuring the lengths of single-stranded RNA separated through denaturing urea polyacrylamide gel electrophoresis (urea PAGE), which are visualized by a label-free fluorescence SYBR Gold, and quantified in a multi-function imager. This label-free method is easy to conduct, has low cost, and lacks the hazard of the traditional radio-labeled method. This method can also be adapted to the other Dicers and small RNAs.
- Published
- 2022
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