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Recommendations for Successful siRNA Library Screens

noted that targeting specific genes caused an approximately four-fold difference in cell number three days post-transfection (Figure 5). While this difference is not critical for microscopic assays and assays that measure phenotypes for individual cells, it is essential that results be normalized to cell number when using ELISAs, enzymatic activity assays, and other assays that measure total signal in a well.

Figure 5. Cell Number Differences in Cells Transfected with Distinct siRNAs. Panel A . HeLa cells (5,000 per well in 96-well plates) were transfected using siPORT NeoFX (Ambion) in triplicate with three different siRNAs targeting each of 59 different kinase-specific siRNAs. Three days post-transfection, cells were fixed and counted using the ArrayScan V (Cellomics). The average number of cells per view field for wells transfected with the various gene-specific siRNAs were plotted. An approximately four-fold variability in cell number in wells transfected with different siRNAs suggests that results from well-based assays (e.g. ELISA, enzymatic activity assay, reporter signal measurements) must be normalized by the number of cells in each well. Panel B. HeLa cells were transfected as described in Panel A. Caspase 3 activity was monitored as described in Figure 3. Green arrows note genes that fell below the selection threshold irrespective of whether normalization was used; yellow arrows note genes that fell below only when normalized by cell number; and red notes the gene that shifted above the selection threshold when normalized to cell number.


(5) Note Cell Type Differences

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