Single siRNAs vs. siRNA Pools: Results
To minimize experimental variability, the data comparing the effects of single and pooled siRNAs on proliferation and caspase 3 activation were generated with the same cell line and substrates at the same time. The results of the screens were used to determine the effectiveness of transfecting a series of three single siRNAs versus transfecting a pool of the three siRNAs for identifying gene "hits." The definition of a hit in our screen was a gene for which two single siRNAs generated 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 complement of interesting genes within the collection being analyzed (Figure 5).
Phenotype vs. siRNA Efficacy
One possible explanation for the discrepancy between single siRNAs and pooled siRNAs is that the levels of target gene expression following transfection were different in each case. To address this, we measured cell number and mRNA knockdown in cells transfected with single and pooled siRNAs targeting nine different genes. As shown in Figure 6, the average correlation between cellular phenotype and mRNA knockdown for each of the different genes was low, indicating that the variability in phenotypes induced by different siRNAs was unlikely to have been caused by siRNA efficacy.
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Figure 6. Phenotype vs. mRNA Knockdown in Transfected Cells. HeLa cells were transfected with three different single siRNAs as well as an siRNA pool targeting each of the nine genes shown on the x-axis. Three days post-transfection, the wells were assayed for cell number (A) using alamarBlue® (AccuMed International). The RNA in the cells was isolated and assayed for target mRNA expression by real-time RT-PCR (B). The alamarBlue and the target mRNA quantification are presented as percentages of cells transfected with with Silencer® Negative Control #1 siRNA (Ambion). |
Conclusions
Similar comparisons of single siRNAs versus pooled siRNAs were carried out using nine different phenotypic assays including protein activation, cell morphology, and cytoskeleton formation. Our results demonstrated that experiments using pooled siRNAs consistently yielded false positive rates of >50% and false negative rates >40%. Testing a single siRNA per target had slightly lower, though no less onerous, rates of false positives and false negatives. The phenotypic differences between single siRNAs and pooled siRNAs targeting the same gene likely reflect the variability in off-target effects of siRNAs. To overcome this, we found it extremely important to use several individual siRNAs per target gene in individual transfections to confirm that a phenotype was specific to the reduction of gene expression and not due to off-target effects of a single siRNA or an siRNA pool.
Scientific Contributors
David Brown, Mike Byrom, Joe Krebs, Kevin Kelnar, Rich Jarvis, Amanda Campbell, Lance Ford • Ambion, Inc.
