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Recommendations for Successful siRNA Library Screens

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(3) Use Individual, Validated siRNAs, Not siRNA Pools

siRNA design algorithms have essentially eliminated the need to use pools of low potency siRNAs. To address whether siRNA pools benefit screening experiments, (sets of) three siRNAs targeting each of 59 kinases were each transfected into HeLa cells sequentially, one by one, and as a pool (Figure 3).

Figure 3. siRNA Pools vs. Single siRNAs. The expression levels of 59 kinases were reduced in individual HeLa cell populations in 96 well plates by transfecting, in triplicate, three single siRNAs (Panel A; #1, #2, #3) as well as a pool of siRNAs (Panel B; #1+#2+#3) targeting each kinase. After assaying cell number with Alamar Blue (Biosource) caspase 3 assays were performed as described for Figure 2. Yellow arrows indicate results that were confirmed targets (i.e. inhibiting kinase activity via siRNA inhibits activation of the apoptosis pathway as measured by caspase 3 activity) by both methods. Red arrows indicate false negative results from pooled transfections, and green arrows indicate false positive results from pooled transfections. The graph (Panel C) shows representative examples of genes identified as positive results, false negative from pooled assays, false positive from pooled assays, and negative results. The yellow line indicates the 70% threshold used to determine "hits."


The transfected cells were monitored for their ability to activate caspase 3 following induction of apoptosis. "Hits" were defined as cells that had at least 30% less caspase 3 activity than negative c
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