In order to fully understand the biological impact of reducing expression of a target gene, it is necessary to correlate the observed phenotype with the level of knockdown induced. Time course and siRNA titration experiments are particularly useful for this endeavor.

The time course of siRNA silencing of survivin mRNA and protein correlated well with the time frames where decreased cell survival/proliferation; changes in nuclear and plasma membrane shape, chromosomal condensation and fragmentation; and phosphatidyl serine externalization were observed. These observations are summarized in the chart in Figure 9 and strongly suggest that specific silencing of survivin by siRNA in HeLa cells is sufficient to cause an increase in apoptosis in the absence of additional apoptotic insults. Our observations are consistent with other investigations on the role of survivin in cell biology and regulation of apoptosis [1].

Figure 9. Correlating Survivin Silencing with Apoptosis Parameters. Data from the experiments in Figure 7-11 is summarized here showing the relationship between silencing survivin over time and the phenotypic effects assayed over this same time frame.

Conclusions
Overexpression of survivin is rapidly becoming a diagnostic and prognostic marker for malignant cancers. Its restricted expression in malignant cells makes it a promising target for novel therapies for cancer treatment. To fully exploit survivin as a target for cancer therapy, we need to more completely understand how survivin inhibits apoptosis, and to identify the biological mechanisms that are critical for its inhibition. We, along with others, have demonstrated that siRNA mediated silencing of survivin in cell culture models is a convenient and efficacious model to understand survivin function. Cell culture is exquisitely sensitive to knockdown by siRNA which will facilitate more in-depth research into this important biological pathway.

Success is maximized when optimized reagents are used. Applied Biosystems, now including Ambion, provides the most complete suite of validated, quality reagents for RNA interference experiments available for this purpose.

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References
1. Carvalho A, Carmena M, Sambade C, Earnshaw WC, Wheatley SP. (2003) J Cell Sci. 116(14): 2987–2998. Epub 2003 Jun 3.