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Updated: Aug 11, 2026

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay
Published on: May 25, 2015
3' UTR seed matches, but not overall identity, are associated with RNAi off-targets
Amanda Birmingham1, Emily M Anderson, Angela Reynolds
1Dharmacon Research, 2650 Crescent Drive, #100, Lafayette, Colorado 80026, USA.
Abstract:
Off-target gene silencing can present a notable challenge in the interpretation of data from large-scale RNA interference (RNAi) screens. We performed a detailed analysis of off-targeted genes identified by expression profiling of human cells transfected with small interfering RNA (siRNA). Contrary to common assumption, analysis of the subsequent off-target gene database showed that overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA, except for near-perfect matches. Instead, off-targeting is associated with the presence of one or more perfect 3' untranslated region (UTR) matches with the hexamer or heptamer seed region (positions 2-7 or 2-8) of the antisense strand of the siRNA. These findings have strong implications for future siRNA design and the application of RNAi in high-throughput screening and therapeutic development.
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