Unit

UNIT 26.4 Cloning of Small RNA Molecules

  1. Sébastien Pfeffer,
  2. Mariana Lagos-Quintana,
  3. Thomas Tuschl

Published Online: 1 NOV 2005

DOI: 10.1002/0471142727.mb2604s72

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Pfeffer, S., Lagos-Quintana, M. and Tuschl, T. 2005. Cloning of Small RNA Molecules. Current Protocols in Molecular Biology. 72:26.4.1–26.4.18.

Author Information

  1. The Rockefeller University, New York, New York

Publication History

  1. Published Online: 1 NOV 2005
  2. Published Print: OCT 2005

Abstract

Small RNAs that are derived from dsRNA precursors act as guide RNAs during sequence-specific epigenetic regulation of eukaryotic gene expression. These small regulatory RNAs are between 20 and 30 nucleotides in length, and fall into one or more of the following categories: small interfering RNAs (siRNAs), microRNAs (miRNAs), and heterochromatic siRNAs (hsiRNAs). Procedures to record the profile of small RNAs expressed in cultured cells or tissues are described. The small RNAs are directionally cloned after isolation from total RNA. The methods rely on T4 RNA ligase-based joining of adapter oligonucleotides to the 3′ and 5′ termini of the pool of small RNAs. The ligation products are reverse transcribed and PCR-amplified. It is recommended to directionally concatamerize the relatively short PCR products before cloning in order to increase the number of RNA sequences obtained per clone.

Keywords:

  • RNAi;
  • miRNA;
  • siRNA;
  • non-coding RNA;
  • cDNA library