Identifying siRNA-induced off-targets by microarray analysis.
RNA interference (RNAi) is an endogenous gene regulatory pathway that the research community has adopted to facilitate the creation of a functional map of the human genome. To achieve this, small interfering RNAs (siRNAs), short synthetic duplexes having complete homology to the intended target, are introduced into cells to silence gene expression via a posttranscriptional cleavage mechanism. While siRNAs can be designed to effectively knock down any target gene, recent studies have shown that these small molecules frequently trigger off-target effects. These unintended events can have a significant impact on experimental outcomes and subsequent data interpretation. As RNAi is envisioned to play a central role in developing a functional map of the human genome, the development of reliable protocols for identifying off-targeted genes is essential. This chapter focuses on the underlying features of siRNA-mediated off-targeting and the state-of-the-art methodology used to identify off-targeted genes via microarray-based gene expression analysis. Future adoption of standards in this field will allow a clean distinction between sequence-specific off-target gene regulation and other forms of gene modulation resulting from delivery effects and other events unrelated to the RNAi pathway.
Anderson
Emily
E
Dharmacon, ThermoFisher Scientific, Lafayette, CO, USA.
Boese
Queta
Q
Khvorova
Anastasia
A
Karpilow
Jon
J
D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D046228:Microarray Analysis / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D035683:MicroRNAs; D034741:RNA, Small Interfering