An example in which the phenotypic effects of an siRNA were correlated with the extent of mRNA knockdown induced is shown in Figure 5. A Silencer Validated siRNA targeting survivin mRNA was transfected into cells. Silencer Negative Control #1 siRNA was transfected into another set of cells. qRT-PCR using a TaqMan Gene Expression Assay showed that the survivin siRNA reduced survivin mRNA levels in these cells by 80% compared to cells treated with the negative control siRNA (Figure 5). In addition, immunofluorescence analysis of survivin protein levels indicated that protein levels were reduced 76% compared to cells transfected with the negative control siRNA (data not shown). In the survivin siRNA treated samples, distinct changes in nuclear morphology, consistent with changes that would be expected for cells undergoing apoptosis, were noted. No distinguishable change in nuclear morphology was noted in cells treated with the negative control siRNA as compared to nontransfected cells. From these data, it can be inferred that knockdown of survivin induces apoptosis in these samples. The next experimental step would be to confirm the results with a second siRNA targeting survivin, and to monitor apoptosis by additional assays (e.g., caspase activity assay, annexin V assay, etc.), while continuing to monitor the extent of survivin mRNA knockdown by qRT-PCR.
Figure 5. Knockdown
of Survivin with a Silencer Validated siRNA and Verification
with TaqMan Gene Expression Assays. HeLa
cells were transfected with 30 nM of a Silencer Validated siRNA