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Designing Controls for siRNA ,,, Experiments


Because of its apparent specificity, reproducibility, and ease of use, RNAi technology is greatly accelerating the functional characterization of disease-relevant genes for drug discovery, target validation, and basic research efforts. To ensure the validity of RNAi data, however, proper experimental design and controls are needed. This article outlines some of the critical parameters for performing RNAi experiments in mammalian cultured cells.


Tips for Experimental Design

Carefully Choose Your siRNA Sequence for Maximum Specificity.
Although data indicate that siRNAs are highly specific, it is important to minimize the possibility of off-target effects by designing siRNAs that have limited sequence similarity to genes other than their intended target. Currently, it is not clearly understood how the number and location of mismatches between the antisense siRNA strand and the target mRNA affect silencing. Some reports indicate that a single mismatch in the center of an siRNA sequence can abolish silencing effects (1,2). In fact, this remarkable specificity has been shown in a model system to permit allele specific gene silencing (2). In contrast, another report indicates that siRNAs with regions of 14-15 contiguous bases of sequence similarity between the siRNA and an mRNA can induce silencing (3). Until we develop a better understanding of the specificity of siRNAs for their
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