Kinetic Analysis of Small Silencing RNA Production by Human and Drosophila Dicer Enzymes In Vitro

Methods Mol Biol. 2018;1680:101-121. doi: 10.1007/978-1-4939-7339-2_7.


Dicer enzymes produce small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), which then are loaded into Argonaute proteins and act as sequence-specific guides. A powerful tool to understand the molecular mechanism of small silencing RNA production by Dicers is an in vitro RNA processing assay using recombinant Dicer proteins. Such biochemical analyses have elucidated the substrate specificities and kinetics of Dicers, the mechanism by which the length of small RNAs produced by Dicers is determined, and the effects of Dicer-partner proteins and endogenous small molecules such as ATP and inorganic phosphate on small RNA production by Dicers, among others. Here, we describe methods for in vitro small RNA production assay using recombinant human and Drosophila Dicer proteins.

Keywords: Dicer; Kinetics; RNA silencing; miRNA; siRNA.

Publication types

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

MeSH terms

  • Animals
  • Drosophila / genetics*
  • Drosophila / metabolism*
  • Humans
  • In Vitro Techniques
  • Isotope Labeling
  • Kinetics
  • RNA Interference*
  • RNA, Small Interfering / genetics*
  • RNA, Small Interfering / metabolism
  • Recombinant Proteins
  • Ribonuclease III / metabolism*


  • RNA, Small Interfering
  • Recombinant Proteins
  • Ribonuclease III