Cloning Argonaute-associated small RNAs from Caenorhabditis elegans

Methods Mol Biol. 2011;725:251-80. doi: 10.1007/978-1-61779-046-1_17.

Abstract

Small RNA pathways fulfill a plethora of gene-regulatory functions in a variety of organisms. In the nematode worm, Caenorhabditis elegans, a number of endogenous small RNA pathways have been described, including the microRNA pathway, the 21U/piRNA pathway, the 26G-RNA pathways, and the 22G-RNA pathways. Argonaute proteins are key effector molecules of each pathway that, together with their small RNA cofactors regulate various processes including developmental timing, fertility, transposon silencing, and chromosome segregation. Although several of the 26 Argonautes in the worm have been studied to date, a number have yet to be fully characterized or their small RNA binding complement defined. The identification of small RNAs that copurify with an Argonaute family member is central to understanding the targets and assessing the function of that Argonaute. Here we discuss the rationale for generating reagents to immunoprecipitate Argonaute complexes and provide a cohesive protocol for the cloning and Illumina deep-sequencing of Argonaute-associated small RNAs in C. elegans.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / immunology
  • Antibodies / metabolism
  • Caenorhabditis elegans / genetics*
  • Caenorhabditis elegans / growth & development
  • Caenorhabditis elegans / metabolism
  • Cloning, Molecular*
  • Epitopes / immunology
  • Epitopes / metabolism
  • Eukaryotic Initiation Factors / immunology
  • Eukaryotic Initiation Factors / metabolism*
  • Gene Library
  • Immunoprecipitation
  • MicroRNAs / genetics*
  • MicroRNAs / isolation & purification
  • MicroRNAs / metabolism
  • Quality Control
  • Reproducibility of Results

Substances

  • Antibodies
  • Epitopes
  • Eukaryotic Initiation Factors
  • MicroRNAs