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Are siRNA Pools Smart?

ated a phenotype that was statistically different from the negative control (Silencer Negative Control #1 siRNA, Ambion) transfected samples in the screening experiment.

False Positives . For both proliferation and caspase 3 assays, less than 60% of the hits predicted by the pooled siRNA results could be validated by the single siRNAs (False Positives, Figure 5).

False Negatives. While the analysis of false positives wastes time and resources, of greater concern were the genes that failed to register as hits when the gene target did indeed participate in the pathway being analyzed. These occurrences, referred to as false negatives, were especially prevalent for the siRNA pools: Results from pooled siRNAs suggested that the gene was not involved in the apoptosis pathway, whereas results from two or more individual siRNAs to the same target suggested the gene was important for activation of the apoptosis pathway. In both screens, the pooled siRNA experiments had a 50% false negative rate, indicating that screening with siRNA pools can result in lost opportunities for target gene identification.

Use of more than one individual siRNA. Though less problematic than experiments using siRNA pools, studies involving only a single gene-specific siRNA were prone to the relatively high rates of false positives and false negatives described above. The use of two, and preferably three, distinct siRNAs per gene significantly decreased the false negative rates of screening, making it possible to identify a more complete c
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