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Recommendations for Successful siRNA Library Screens

tive rates that exceed 40% (data not shown). As seen in Figure 4, the false positive and false negative rates associated with using a single siRNA per target gene (#1, #2, #3) are lower than when siRNA were pooled. To minimize the number of False Negative (missed) genes and the number of False Positive (not confirmed by other specific siRNAs) genes, researchers should analyze the combined results from separate transfections of three different gene-specific siRNAs (#1+#2+#3). For larger screening experiments where this may not be feasible, results from single siRNA transfections of one (#1, #2, #3) or two (#1+#2, #1+#3, #2+#3) gene-specific siRNA still reduced the number of missed or False Positive genes compared to transfection of pools of siRNAs.

Figure 4. Screening With Individual siRNAs Results in Fewer False Positives and False Negatives Than Screening With Pooled siRNAs . Inhibition of 11 of 59 kinase genes via transfection of siRNAs inhibited etoposide-activated apoptosis (monitored by caspase 3 activity, see Figure 3). By definition, repression of Confirmed Positive genes resulted in low caspase 3 activity in assays from at least two gene-specific siRNAs. To examine the accuracy of screening methods for identifying important genes, we analyzed the results of transfection experiments involving either a mixture of three siRNAs that target the same gene (siRNA pool) or one of the three siRNAs (#1, #2, #3).


(4) Correct for Cell Number

Inhibition of genes critical for cell division or cell survival can greatly affect cell number. For example, using a series of kinase-specific siRNAs for preliminary work, we
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