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In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo
Author(s) -
Rao M.,
Baraban J.H.,
Rajaii F.,
Sockanathan S.
Publication year - 2004
Publication title -
developmental dynamics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.634
H-Index - 141
eISSN - 1097-0177
pISSN - 1058-8388
DOI - 10.1002/dvdy.20161
Subject(s) - rna interference , biology , gene silencing , electroporation , small interfering rna , rna silencing , trans acting sirna , in ovo , microbiology and biotechnology , gene expression , piwi interacting rna , rna , gene , rna induced silencing complex , genetics , embryo
The combination of emergent RNA interference (RNAi) technology with in ovo electroporation in the chick embryo has the potential to provide a powerful and rapid means for functional analyses of novel genes in vivo. In this study, we show that electroporation of short 21‐bp RNA duplexes (siRNAs) is a quick and simple method for silencing exogenous and endogenous gene expression in vivo. Quantitative comparisons with two other RNAi protocols that use long double‐stranded RNA duplexes and endonuclease‐digested duplexes (esiRNAs) demonstrate that siRNAs are significantly more effective at reducing gene expression. Furthermore, we also find that much higher amounts of siRNA are required for silencing of endogenous gene expression relative to plasmid‐borne reporter constructs. In short, these results demonstrate that siRNAs are the most effective type of double‐stranded RNA duplex for silencing gene expression and suggest that there might be important differences between silencing endogenous and exogenous genes. Finally, we review the parameters for each of these RNA‐based methods of RNAi and the controls required to analyze RNAi data in the context of the developing vertebrate embryo. Developmental Dynamics 231:592–600, 2004. © 2004 Wiley‐Liss, Inc.