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RNA Interference as a Tool to Selectively Down-Modulate Protein Function

  • Seila Fernandez-Fernandez
  • Juan P. BolañosEmail author
Protocol
  • 924 Downloads
Part of the Neuromethods book series (NM, volume 90)

Abstract

RNA interference (RNAi) is a rapid and efficient technique to cause highly selective protein loss of function in cells. In this chapter, we would like to describe all necessary steps to implement this approach for the first time in a laboratory that is not used to deal with gene databases information and related technical skills. Using either rational criteria or sophisticated—otherwise straightforward—online software, it is now reasonably feasible for everyone to design the small interfering RNA (siRNA) or the short hairpin RNA (shRNA) molecules to knockdown—or “silence”—your protein of interest. We provide recommendations on how to retrieve the mRNA sequence of your protein of interest from the online databases and to identify the nucleotide-sequence target on the mRNA. In addition, we provide clues aimed to predict the selectivity of the target chosen and to obtain a long-lasting knockdown effect by expressing shRNA from a plasmid vector. How to get high transfection efficiencies in hard-to-transfect-cells, such as primary neurons in culture, and how to test the efficacy of the RNAi approach, is also recommended. We expect to encourage researchers to use this now easily accessible technique as an alternative—or additional—approach to the usually unspecific pharmacological inhibition when studying neural metabolism.

Key words

RNA interference Small interfering RNA Short hairpin RNA Transfection methods Transfection efficiency Neurons 

Notes

Acknowledgements

This work was supported by FEDER (European regional development fund), Ministerio de Ciencia e Innovacion (SAF2010-20008 and Consolider-Ingenio CSD2007-00020), and Junta de Castilla y Leon (SA112A12-2).

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© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular Biology, Institute of Functional Biology and Genomics (IBFG)University of Salamanca-CSIC-IBSALSalamancaSpain

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