Screening Target Specificity of siRNAs by Rapid Amplification of cDNA Ends (RACE) for Non-Sequenced Species

Abstract

RNA interference (RNAi) is the process of sequence-specific posttranslational gene silencing triggered by double-stranded RNAs (dsRNAs). RNAi is a widely used approach for studying gene function. However, studies have shown that using siRNA can lead to off-target effects when the siRNA contains sufficient sequence identity to non-target mRNA sequences. One of the important steps in designing dsRNA is verification that it has sequence identity to only the target mRNA. In this report, we propose an approach for primary screening dsRNAs for potential off-target effects by using rapid amplification of cDNA ends. This method can be especially useful for model systems using species that have limited availability of sequence data.

Precise RNAi-mediated silencing of metabolically active proteins in the defence secretions of juvenile leaf beetles

“…These approaches are not suitable for organisms whose genomes have yet to be sequenced. In the last few years, several approaches have been used to detect off targets for those species, such as screening for target specificity by rapidly amplifying cDNA ends [54]. Another approach has used microarrays to compare the cDNAs from treated groups with those from non-treated groups; such comparisons offer proof of differentially expressed transcripts via qPCR [55].…”