Abstract
In plants, small RNA (microRNAs and other endogenous small RNAs)-guided target gene expression is vital for a wide variety of biological processes including adaptation to stress conditions. Identification of stress-regulated microRNAs or other classes of endogenous small RNAs advances our understanding of post-transcriptional gene regulation important for plant stress tolerance. This chapter describes a detailed step-by-step protocol for cloning of small RNAs. Following 5ʹ and 3ʹ adapter ligation to the purified small RNAs, cDNA will be synthesized using reverse transcription, which will be further amplified using a polymerase chain reaction. The resulting small DNA fragments can be either cloned and sequenced using traditional sequencing method or subjected to direct high-throughput pyrosequencing or sequencing-by-synthesis technology.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297.
Addo-Quaye, C., Eshoo, T.W., Bartel, D.P., and Axtell, M.J. (2008) Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Curr Biol 18, 758–762.
Brodersen, P., Sakvarelidze-Achard, L., Bruun-Rasmussen, M., et al. (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320, 1185–1190.
Zhou, X., Wang, G., Sutoh, K., Zhu, J.K., and Zhang, W. (2008) Identification of cold-inducible microRNAs in plants by transcriptome analysis. Biochem Biophys Acta 1779, 780–788.
Lu, S., Sun, Y.H., and Chiang, V.L. (2008) Stress-responsive microRNAs in Populus. Plant J 55, 131–151.
Zhao, B., Liang, R., Ge, L., et al. (2007) Identification of drought-induced micro-RNAs in rice. Biochem Biophys Res Commun 354, 585–590.
Lu, C., Meyers, B.C., and Green, P.J. (2007) Construction of small RNA cDNA libraries for deep sequencing. Methods 43,110–117.
Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. (2001) Identification of novel genes coding for small expressed RNAs. Science 294, 853–858.
Acknowledgments
Support for the research in R.S. laboratory was provided by the Oklahoma Agricultural Experiment Station. Support for CAH and JLR was provided by funding from DGAPA-UNAM (IN-227706) and from CONACyT, Mexico (J48740).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press
About this protocol
Cite this protocol
Reyes, J.L., Arenas-Huertero, C., Sunkar, R. (2010). Cloning of Stress-Responsive MicroRNAs and other Small RNAs from Plants. In: Sunkar, R. (eds) Plant Stress Tolerance. Methods in Molecular Biology, vol 639. Humana Press. https://doi.org/10.1007/978-1-60761-702-0_14
Download citation
DOI: https://doi.org/10.1007/978-1-60761-702-0_14
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-701-3
Online ISBN: 978-1-60761-702-0
eBook Packages: Springer Protocols