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Recommendations for Successful siRNA Library Screens

educe siRNA Concentration

Although many siRNA transfections are still performed with 100 nM siRNA, published results indicate that transfecting lower siRNA concentrations can reduce nonspecific effects exhibited by some siRNAs [1]. Silencer siRNAs reduce target mRNA levels when transfected at significantly lower concentrations (Figure 1). To confirm that phenotypes induced by 100 nM siRNA were apparent at lower siRNA concentrations, cells were transfected with multiple concentrations of five different siRNAs found to block caspase 3 activation (a marker for apoptosis) in a phenotypic screen. As predicted by mRNA studies, there was essentially no difference in the phenotype induced by the Silencer Pre-designed siRNAs even when very low siRNA concentrations (10 nM) were used for transfection (Figure 2).

Figure 1. siRNA Concentration vs. mRNA Knockdown. HeLa cells were transfected with 1-100 nM of six different Silencer Pre-designed siRNAs (Ambion). siRNAs transfected at 1-3 nM concentrations often elicited effective silencing .

Figure 2. Low siRNA Concentrations are Effective in Functional Assays. HeLa cells were transfected with siRNA to nine different targets. One day post-transfection, cells were treated with etoposide to induce apoptosis. Three days post-transfection, cells were lysed and monitored for caspase 3 activity. Results for each well are graphed relative to the average caspase 3 activity from a negative control transfected s
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