Enzymatic production of small interfering RNAs

doi:10.1017/CBO9780511546402.012

9 - Enzymatic production of small interfering RNAs

Published online by Cambridge University Press: 31 July 2009

Muhammad Sohail and

Graeme Doran

Edited by

Krishnarao Appasani

Foreword by

Andrew Fire and

Marshall Nirenberg

Show author details

Muhammad Sohail: Affiliation:
MRC Research Associate, University of Oxford, Department of Biochemistry
Graeme Doran: Affiliation:
Department of Human Anatomy and Genetics, University of Oxford
Krishnarao Appasani: Affiliation:
GeneExpression Systems, Inc., Massachusetts
Andrew Fire: Affiliation:
Stanford University, California

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Summary

Introduction

RNA interference is a powerful natural phenomenon of post-transcriptional gene silencing that has been found in several biological systems. Small interfering RNAs (siRNAs) are important reagents in the RNA interference pathways that determine gene-specificity of the pathway. RNA interference can be induced in a system by the application of artificial siRNAs. Small interfering RNAs are vital intermediates in natural RNA interference that determine gene specificity of the phenomenon. RNA interference can be engineered for a gene for which this process does not occur in nature by applying artificial siRNAs, to study its function or for therapeutic purpose (Dykxhoorn et al., 2003). The original way of producing siRNAs was by chemical synthesis, which is expensive and usually there is no guarantee that reagents would produce desired gene silencing effects. A number of laboratories have developed alternative methods of producing siRNAs involving plasmid- or virus-mediated intra cellular expression and in vitro with the use of bacterial or viral enzymes. This chapter provides an overview of methods of producing siRNAs by enzymatic means.

Production of siRNAs by in vitro transcription

Donze and Picard (2002) reported a method based on the use of T7 RNA polymerase and short synthetic oligonucleotides as template (Milligan et al., 1987) in producing siRNAs and showed that siRNAs produced with this in vitro transcription method were capable of silencing GFP expression in human HeLa and that of protein kinase PKR in HEK293T cells.

Type: Chapter
Information: RNA Interference Technology
From Basic Science to Drug Development
, pp. 139 - 144

DOI: https://doi.org/10.1017/CBO9780511546402.012 [Opens in a new window]

Publisher: Cambridge University Press

Print publication year: 2005

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