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Identification of microRNAs and Other Tiny Noncoding RNAs by cDNA Cloning

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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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© 2004 Humana Press Inc.

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Ambros, V., Lee, R.C. (2004). Identification of microRNAs and Other Tiny Noncoding RNAs by cDNA Cloning. In: Gott, J.M. (eds) RNA Interference, Editing, and Modification. Methods in Molecular Biology, vol 265. Humana Press. https://doi.org/10.1385/1-59259-775-0:131

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  • DOI: https://doi.org/10.1385/1-59259-775-0:131

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-242-1

  • Online ISBN: 978-1-59259-775-8

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