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Assessing Gene Function with siRNA Libraries

48 hours after siRNA transfection as monitored by real-time PCR. For each kinase siRNA, target mRNA levels were reduced by 70% or more as compared to the levels of mRNA obtained after transfection with the negative control siRNA.

Figure 1. mRNA Silencing by 178 Kinase siRNAs from the Silencer Kinase siRNA Library. HeLa cells were plated at approximately 8000 cells per well in 96 well plates. Twenty-four hours later the cells were transfected in triplicate with each siRNA (100 nM). Forty-eight hours post-transfection, RNA was isolated, converted to cDNA, and analyzed by real-time PCR. Shown are the relative mRNA levels compared to cells transfected with a control scrambled siRNA (red bar, Silencer Negative Control #1 siRNA).


Effects on Cell Proliferation
Figure 2 demonstrates the effect of each siRNA on cell proliferation. In wells in which the negative control siRNA was transfected, there were approximately 1,250 cells/microscopic image after 48 hours (Figure 2). Inhibition of cyclin B1, which is known to be critical for initiation of mitosis, results in growth arrest. As expected, cell proliferation was dramatically inhibited by the siRNA targeting cyclin B1; there were <700 cells/microscopic image in wells transfected with this siRNA. Figure 2 also shows several kinase siRNAs that inhibit cell proliferation. This inhibition can result from a number of underlying causes that fall into three broad categories: effects causing cell necrosis, effects causing apoptosis, and effects causing cell cycle deregulation. Interestingly, a few kinase siRNAs appear to have a stimulatory effect on cell proliferation.

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