Abstract
MicroRNAs are short single-stranded RNA molecules (18–25 nucleotides). Because of their ability to silence gene expressions, they can be used to diagnose and treat tumors. Experimental construction of microRNA libraries was the most important step to identify microRNAs from animal tissues. Although there are many commercial kits with special protocols to construct microRNA libraries, this chapter provides the most reliable, high-throughput, and affordable protocols for microRNA library construction. The high-throughput capability of our protocols came from a double concentration (3 and 15%, thickness 1.5 mm) polyacrylamide gel electrophoresis (PAGE), which could directly extract microRNA-size RNAs from up to 400 μg total RNA (enough for two microRNA libraries). The reliability of our protocols was assured by a third PAGE, which selected PCR products of microRNA-size RNAs ligated with 5′ and 3′ linkers by a miRCatTM kit. Also, a MathCAD program was provided to automatically search short RNAs inserted between 5′ and 3′ linkers from thousands of sequencing text files.
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Acknowledgment
All the experimental data were obtained from Dr. Jianbo Yao’s Laboratory of Animal Biotechnology and Genomics, Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV 26506-6108, USA. Dr. Yao had read the manuscript and gave a permit to publish the paper without his name as a coauthor.
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Xiao, C. (2011). High-Throughput and Reliable Protocols for Animal MicroRNA Library Cloning. In: Wu, W. (eds) MicroRNA and Cancer. Methods in Molecular Biology, vol 676. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-863-8_10
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DOI: https://doi.org/10.1007/978-1-60761-863-8_10
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-862-1
Online ISBN: 978-1-60761-863-8
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