A rapid and sensitive assay for quantification of siRNA efficiency and specificity
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Last Update: 2020-11-24
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Source: Internet
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Author: User
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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.
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