title: A rapid and sensitive assay for quantification of siRNA efficiency and specificity.
abstract: RNA Interference has rapidly emerged as an efficient procedure for knocking down gene expression in model systems. However, cross-reactivity, whereby multiple genes may be simultaneously targeted by a single short interfering RNA (siRNA), can potentially jeopardize correct interpretation of gene function. As such, it is essential to test the specificity of a siRNA prior to a full phenotypic analysis. To this end, we have adapted a reporter-based assay harnessing the sensitivity of luciferase activity to provide a quantitative readout of relative RNAi efficacy and specificity. We have tested different siRNAs directed against Thymosin beta4 (Tbeta4); determined their effectiveness at silencing Tbeta4 and have both excluded off-target silencing of the Tbeta4 homologue Thymosin beta10 (Tbeta10) and demonstrated partial knockdown of Tbeta10 despite significant (12/23; 52%) sequence mismatch. This assay system is applicable to any RNAi study where there is a risk of targeting homologous genes and to the monitoring of off-target effects at the genome level following microarray expression profiling.
authors.affiliation: Molecular Medicine Unit, Institute of Child Health. 30 Guilford Street, London WC1N 1EH. United Kingdom.
authors.forename: Nicola
authors.initials: N
authors.lastname: Smart
authors.forename: Peter James
authors.initials: PJ
authors.lastname: Scambler
authors.forename: Paul Richard
authors.initials: PR
authors.lastname: Riley