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Quantification of dicer activity in mammalian cell lysates using a non-radioactive fluorescence method

Anna Emde, Natalia Rivkin, Mark A. Behlke, Eran Hornstein

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

In this chapter, we provide a protocol for design and usage of an in vitro, cell-free Dicing assay. Our assay is based on previously established methods and improves the ability to quantitatively measure the catalytic efficacy of the Dicer complex under various cellular conditions via non-radioactive, emitted fluorescence signal readout. Because radioactive labeling is substituted by measuring fluorescent signal the method is easy to handle. Additionally, the use of a defined amount of Cy3-quenched double-stranded substrate allows for precise detection of small changes in cellular Dicing complex activity. This experimental approach might be valuable for investigating cellular miRNA biogenesis and activity under various conditions in health and disease.

Original languageEnglish
Title of host publicationMicroRNA Technologies
EditorsMin Jeong Kye
PublisherHumana Press
Chapter7
Pages21-27
Number of pages7
DOIs
StatePublished - 15 Nov 2016

Publication series

NameNeuromethods
Volume128
ISSN (Print)0893-2336

All Science Journal Classification (ASJC) codes

  • General Neuroscience
  • General Biochemistry,Genetics and Molecular Biology
  • General Pharmacology, Toxicology and Pharmaceutics
  • Psychiatry and Mental health

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