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Identification of microRNAs and Other Tiny Noncoding RNAs by cDNA Cloning

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 265)

Abstract

MicroRNAs (miRNAs) and other small RNAs can be identified by cloning and sequencing cDNAs prepared from the ∼22-nt fraction of total RNA. Methods are described for the construction of cDNA libraries from small noncoding RNAs through the use of T4 RNA ligase, reverse transcriptase, and polymerase chain reaction. cDNAs are cloned in λ or plasmid vectors, and the sequences are compared to annotated genomic sequence databases, and analyzed by RNA folding programs to distinguish miRNA sequences from other small RNAs of similar size. Northern blot hybridization is used to confirm the expression of small RNAs in vivo.

Key Words

microRNA noncoding RNA RNA ligase SMART RNA folding cDNAs Northern blot 

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Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  1. 1.Department of GeneticsDartmouth Medical SchoolHanover

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