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Cloning of Stress-Responsive MicroRNAs and other Small RNAs from Plants

 Last Update: 2020-11-20
 Source: Internet
 Author: User

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pyrosequencing

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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, c
DNA
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.

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