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Methods Mol Biol. 2011;744:13-25. doi: 10.1007/978-1-61779-123-9_2.

Caveat of RNAi in plants: the off-target effect.

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Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK, USA.


RNA interference (RNAi), mediated by short interfering RNAs (siRNAs), is one of the widely used functional genomics method for suppressing the gene expression in plants. Initially, gene silencing by RNAi mechanism was believed to be specific requiring sequence homology between siRNA and target mRNA. However, several recent reports have showed that non-specific effects often referred as off-target gene silencing can occur during RNAi. This unintended gene silencing can lead to false conclusions in RNAi experiments that are aimed to study the functional role of a particular target gene in plants. This especially is a major problem in large-scale RNAi-based screens aiming for gene discovery. Hence, understanding the off-target effects is crucial for minimizing such effects to better conclude gene function analyzed by RNAi. We discuss here potential problems of off-target gene silencing and focus on possibilities that favor this effect during post-transcriptional gene silencing. Suggestions to overcome the off-target effects during RNAi studies are also presented. We believe that information available in present-day plant science literature about specificity of siRNA actions is inadequate. In-depth systematic studies to understand their molecular basis are necessary to enable improved design of more specific RNAi vectors. In the meantime, gene function and phenotype results from present-day RNAi studies need to be interpreted with caution.

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