Cell Proliferation Results
Cell number was measured using an alamarBlue® (AccuMed International) microplate-based assay. These results were confirmed by high-throughput microscopy. As shown in Figure 3A, siRNAs targeting the various kinases differentially affected cell number relative to negative control transfected samples. Different siRNAs targeting the same gene typically had similar effects on cell proliferation; however, it was common for one of the three single siRNAs to behave differently from the other two siRNAs. Similarly, siRNA pools often failed to display a phenotype consistent with the majority of the siRNAs that were individually transfected (Figure 3B).
Apoptosis Assay Results
During screens for kinases that influence apoptosis, both cell number and caspase 3 activity were measured following siRNA transfection and etoposide treatment. Assaying for cell number was necessary because some of the transfected siRNAs differentially altered cell growth or survival. Normalization of the caspase 3 activity data by cell number allowed us to express data as caspase 3 activity per cell. As expected, reducing expression of several kinases inhibited activation of caspase 3 by etoposide, suggesting that these kinases play a role in apoptosis (Figure 4). Consistent with the cell proliferation results, the single siRNAs for each gene had similar, though non-identical effects, on caspase 3 activity. Also consistent with the cell proliferation results, the siRNA pools only partially overlapped the results of the single siRNAs (Figure 5). Examples of single and pooled siRNA results are shown in Figure 4B.
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Figure 5. Overall Performance of Single siRNAs and siRNA Pools. Using the definition of a hit as a gene for which two single siRNAs produce a phenotype that is significantly different than samples transfected with a negative control siRNA, the performance of each of the single siRNAs (siRNA #1, #2, or #3) as well as the siRNA pool of the three siRNAs was compared. The combined results of screening with two (#1+#2, #1+#3, and #2+#3) or three (#1+#2+#3) individual siRNAs illustrates the higher accuracy of this strategy. Accurate predictions of hits and inaccurate predictions of hits are shown in green and orange, respectively. False negatives are shown in grey. |
