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.
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).
Similar comparisons of single siRNAs versus
pooled siRNAs were carried out using nine different phenotypic
assays including protein activation, cell mor