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.
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 . Three
days post-transfection, the cells were assayed using alamar