Screening with small interfering RNAs (siRNAs) is an important method for identifying genes involved in biological pathways. This article analyzes the effectiveness of two strategies--transfection of individual siRNAs and transfection of pools of siRNAs--for large-scale screening experiments. The increased effectiveness of testing at least two distinct, algorithm-derived siRNAs to the same target results in decreased use of resources required to screen false positive targets, and more importantly, minimizes the risk of missing valid targets.
In initial RNA interference studies, many researchers opted to combine multiple small interfering RNAs (siRNAs) that targeted distinct regions of the same gene to facilitate degradation of the target mRNA. The desire to mix siRNAs arose primarily from the finding that less than 50% of random-designed siRNAs significantly reduced target gene expression. These experiments showed that combinations of siRNAs did not appear to function synergistically to affect target gene expression, and on occasion less active siRNAs interfered with the activities of the higher efficacy siRNAs. Nevertheless, the pooling strategy increased the chances of reducing target gene expression of random-designed siRNAs.
The realization that potent siRNAs share sequence characteristics has led to siRNA design algorithms that significantly improve the percentage of effective siRNA sequences. For instance, the Cenix siRNA design algorithm--upon which Ambion's Silencer® Pre-designed siRNAs are based--has a success rate of >80%, with the majority of the siRNAs providing >85% reduction in target gene expression (Figure 1). Given the success rate of individual algorithm-designed siRNAs, the benefits of siRNA pooling have decreased (Figure 2). Furthermore, the recent observations of off-target effects by siRNAs [1] suggest that combining multiple siRNAs might actually increase the chances that genes other than the target are being affected.
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Figure 1. Efficacy of Silencer® Pre-designed siRNAs. More than 1100 siRNAs targeting nearly 400 endogenously expressed human genes were tested for target mRNA reduction. More than 82% of the siRNAs reduced target gene expression by at least 70% and more than 61% reduced target gene expression by at least 85%. All siRNA designs were based on an algorithm developed by Cenix BioScience. |
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Figure 2. Target mRNA Reduction by Single siRNAs and siRNA Pools. Cells transfected with three individual Silencer® Pre-designed siRNAs (30 nM) or a pool of all three siRNAs (10 nM each) were monitored for target mRNA reduction by real-time RT-PCR 48 h post-transfection. Relative target mRNA expression was taken as a percentage of mRNA expressed in cells transfected with Silencer® Negative Control #1 siRNA (Ambion). All real-time RT-PCR data were normalized with 18S rRNA real-time RT-PCR. |
