A SYBR Gold-based Label-free in vitro Dicing Assay

Qian Wang; Yan Xue; Laixing Zhang; Zhenhui Zhong; Suhua Feng; Changshi Wang; Lifan Xiao; Zhenlin Yang; Jake C Harris; Zhe Wu; Jixian Zhai; Maojun Yang; Sisi Li; Steven E Jacobsen; Jiamu Du

doi:10.21769/BioProtoc.4382

A SYBR Gold-based Label-free in vitro Dicing Assay

Bio Protoc. 2022 Apr 5;12(7):e4382. doi: 10.21769/BioProtoc.4382.

Authors

Qian Wang¹, Yan Xue², Laixing Zhang³, Zhenhui Zhong², Suhua Feng², Changshi Wang¹, Lifan Xiao¹, Zhenlin Yang¹, Jake C Harris⁴, Zhe Wu¹, Jixian Zhai¹, Maojun Yang³, Sisi Li⁵, Steven E Jacobsen^{2

6}, Jiamu Du¹

Affiliations

¹ Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes, Institute of Plant and Food Science, School of Life Science, Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China.
² Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA.
³ Ministry of Education Key Laboratory of Protein Science, Tsinghua-Peking Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
⁴ Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK.
⁵ Department of Biochemistry and Molecular Biology, International Cancer Center, Shenzhen University Health Science Center, Shenzhen, 518060, China.
⁶ Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095, USA.

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.

Keywords: Dicer; Dicing activity; SYBR Gold; Small RNA; Urea PAGE.

Grants and funding

R35 GM130272/GM/NIGMS NIH HHS/United States