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Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing
Published on: November 18, 2014
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A convenient strategy to clone small RNA and mRNA for high-throughput sequencing
Lichao Li1, Hui Dai1, An-Phong Nguyen1
1Department of Molecular, Cell, and Systems Biology, University of California, Riverside, California 92521, USA.
RNA (New York, N.Y.)
|November 23, 2019
Summary
This study introduces a novel, streamlined method for cloning small RNA and mRNA, even with modifications. The efficient, all-liquid-based process requires minimal RNA input and labor, enhancing sensitivity and versatility for various genomic library preparations.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- High-throughput sequencing requires cDNA/DNA library preparation with specific linkers.
- Small RNA cloning is challenging due to modifications (e.g., 5' cap, triphosphate, 2'-O-methyl) and low expression levels, necessitating complex pre-cloning steps.
- Current methods often require separate cloning systems for small RNA and mRNA.
Purpose of the Study:
- To develop a convenient, sensitive, and cost-effective method for cloning 5' modified or unmodified small RNA.
- To create a unified cloning strategy applicable to both small RNA and mRNA.
- To enable genomic library preparation using barcoded PCR primers.
Main Methods:
- An all-liquid-based reaction system performed in a single PCR tube.
- Utilizes as little as 20 ng of total RNA for cloning.
- A 7-hour cloning process with approximately 1 hour of hands-on labor.
Main Results:
- Successfully clones 5' modified or unmodified small RNA and mRNA.
- Demonstrates high sensitivity, versatility, and cost-effectiveness compared to existing methods.
- Compatible with barcoded PCR primers for non-cDNA applications, including genomic libraries.
Conclusions:
- The new method simplifies RNA and DNA library preparation, especially for modified small RNAs.
- Offers a more efficient, sensitive, and versatile alternative to traditional cloning techniques.
- The all-liquid-based, automated potential streamlines high-throughput sequencing workflows.
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