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A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools
Published on: August 21, 2019
High-throughput in vitro processing of human primary microRNA by the recombinant microprocessor
Kijun Kim1,2, V Narry Kim1,2
1Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea.
This study presents a high-throughput assay for analyzing microRNA processing efficiency using recombinant Microprocessor complex and deep sequencing. The protocol enables simultaneous mapping of cleavage sites for numerous primary microRNAs (pri-miRNAs).
Area of Science:
- Molecular Biology
- Biochemistry
- Genomics
Background:
- MicroRNAs (miRNAs) are crucial regulators of gene expression.
- Efficient and accurate processing of primary miRNAs (pri-miRNAs) by the Microprocessor complex is essential for miRNA biogenesis.
- Understanding pri-miRNA processing is key to deciphering gene regulation.
Purpose of the Study:
- To develop and describe a high-throughput in vitro assay for assessing pri-miRNA processing.
- To enable simultaneous mapping of cleavage sites and measurement of processing efficiency for a large number of pri-miRNAs.
- To provide a modifiable protocol for investigating factors affecting pri-miRNA processing.
Main Methods:
- Utilized a high-throughput in vitro processing assay with 1,881 human pri-miRNAs.
- Employed recombinant Microprocessor complex for processing.
- Incorporated deep sequencing library generation for data analysis.
- Modified wild-type pri-miRNAs to examine cis-acting elements.
Main Results:
- Established a comprehensive protocol for analyzing pri-miRNA processing.
- Enabled simultaneous mapping of cleavage sites across numerous pri-miRNA substrates.
- Quantified processing efficiency for a large set of human pri-miRNAs.
- Demonstrated the protocol's adaptability for studying regulatory factors and cis-elements.
Conclusions:
- The described protocol offers a powerful tool for high-throughput analysis of pri-miRNA processing.
- This method facilitates large-scale investigation into miRNA biogenesis and regulation.
- The assay is versatile and can be adapted to study chemical or protein effects on pri-miRNA processing.
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