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; C. Harris; Zhe Wu; Jixian Zhai; Maojun Yang; Sisi Li; Steven Jacobsen; Jiamu Du

doi:10.21769/BioProtoc.4382

Bio-Protocol (Apr 2022)

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,
C. Harris,
Zhe Wu,
Jixian Zhai,
Maojun Yang,
Sisi Li,
Steven Jacobsen,
Jiamu Du

Affiliations

Qian Wang: 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
Yan Xue: Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA
Laixing Zhang: 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
Zhenhui Zhong: Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA
Suhua Feng: Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA
Changshi Wang: 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
Lifan Xiao: 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
Zhenlin Yang: 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
C. Harris: Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK
Zhe Wu: 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
Jixian Zhai: 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
Maojun Yang: 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
Sisi Li: Department of Biochemistry and Molecular Biology, International Cancer Center, Shenzhen University Health Science Center, Shenzhen, 518060, China
Steven Jacobsen: Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USAHoward Hughes Medical Institute, University of California, Los Angeles, CA 90095, USA
Jiamu Du: 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

DOI: https://doi.org/10.21769/BioProtoc.4382
Journal volume & issue: Vol. 12, no. 7

Abstract

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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 in Bio-Protocol

ISSN: 2331-8325 (Online)
Publisher: Bio-protocol LLC
Country of publisher: United States
LCC subjects: Science: Biology (General)
Website: https://bio-protocol.org

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