Skip to content


Biological Procedures Online

Open Access

A rapid and sensitive assay for quantification of siRNA efficiency and specificity

  • Nicola Smart1,
  • Peter James Scambler1 and
  • Paul Richard Riley1Email author
Biological Procedures Online7:710001

Received: 14 October 2004

Accepted: 23 December 2004


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 β4 (Tβ4); determined their effectiveness at silencing Tβ4 and have both excluded off-target silencing of the Tβ4 homologue Thymosin β10 (Tβ10) and demonstrated partial knockdown of Tβ10 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.

Indexing terms

Thymosin beta(4)Thymosin beta(10)