Comparing siRNA Pools and Single siRNAs: Experimental Design and Results
Quantitative, phenotypic assays were used to determine how well single siRNAs performed relative to siRNA pools. siRNAs targeting 59 kinases, along with positive and negative control siRNAs, were studied. Cell populations were transfected with three single siRNAs (siRNA #1, siRNA #2, or siRNA #3), all targeting the same gene, or a pool of the three siRNAs (siRNA #1 + siRNA #2 + siRNA #3). In the experiments described below, cell number was monitored three days post-transfection (Figure 3), or apoptosis was induced using etoposide one day after transfection, and caspase 3 activity was assayed three days post-transfection (Figure 4). All transfected cells were assayed on the same day using master mixes of dyes and substrates to minimize experimental variability in the studies.
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Figure 3. Cell Proliferation--Single siRNAs vs. siRNA Pools. (A) Expression levels of 59 kinases were reduced in HeLa cells by transfection in 96 well plates of three individual kinase-specific siRNAs or a pool of the three siRNAs. All samples were performed in triplicate. To each well containing siPORT™ NeoFX™ Transfection Agent and either one of three siRNAs (30 nM) or the three pooled siRNAs (10 nM each), 8x103 HeLa cells were added in a process termed reverse transfection [2]. Three days post-transfection, the cells were assayed using alamarBlue® (AccuMed International). The mean ± standard deviation is presented. The bars in the graph represent the average alamarBlue result for each siRNA or siRNA pool relative to a negative control (Silencer® Negative Control #1 siRNA; Ambion) transfected sample. (B) Representative results from specific genes are presented to exemplify the types of agreement and disagreement that occurred between expression results using the various siRNA combinations. Zero on the y axis is set to 2 standard deviations below the average signal from the negative control transfected samples. Bars that fall below zero are significantly different than the negative control and thus represent hits in our assay. |
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Figure 4. Caspase 3 Activation--Single siRNAs vs. siRNA Pools. (A) The expression levels of 59 kinases were reduced in HeLa cells by transfection of three single kinase-specific siRNAs or a pool of the siRNAs in 96 well plates. All samples were performed in triplicate. One day post-transfection, the cells were treated with etoposide to activate the apoptosis pathway. Three days post-transfection, the cells were counted and lysed. The caspase 3 activity in the lysates was assayed and normalized by cell number. The mean ± standard deviation of the three replicates is presented, where zero on the y axis is set to 2 standard deviations below the average signal from the negative control transfected samples. So bars that fall below zero are significantly different than the negative control and thus represents hits in our assay. The bars in the graph represent the mean caspase 3 activity relative to a negative control (Silencer® Negative Control #1 siRNA; Ambion) transfected sample. (B) Representative results from specific genes illustrate the types of agreement and disagreement that occurred between the various siRNAs. |
