( erent kinases for their exp...)Assessing Gene Function with siRNA Libraries

erent kinases, for their experiment. These siRNAs were individually transfected in triplicate at a final concentration of 100 nM into HeLa cells, plated at approximately 8000 cells per well in 96 well plates 24 hours prior to transfection. A validated siRNA targeting cyclin B1 was used as a positive control. Cyclin B1 is a well-studied cell cycle regulator known to play a key role in initiating mitosis, and therefore was expected to yield a clear decrease in mitotic index in this study. The Silencer Negative Control #1 siRNA (Ambion) was used as a "scrambled" sequence negative control.

Forty eight hours after transfection, cells were fixed and stained with DAPI to reveal chromatin, with antitubulin for microtubule distribution analysis, and with an antiphosphohistone H3 antibody to identify mitotic cells. The extent of cell proliferation was monitored by counting the number of cells in each well. The percentage of cells undergoing mitosis, or 'the mitotic index', was evaluated by fluorescence microscopy. Additionally, RNA was isolated from parallel samples and reverse transcribed to produce cDNA, and then target levels were measured by real-time PCR.

Verifying siRNA Efficacy
An important component of data analysis in any siRNA experiment is to monitor the extent of mRNA degradation, or 'knockdown', elicited by a particular siRNA. Real-time PCR is a rapid and sensitive technique that is ideally suited for this purpose. Figure 1 shows the target mRNA remaining
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