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Assessing Gene Function with siRNA Libraries

en monitored 48 hours after siRNA transfection. Decreases in mitotic index reflect either a shortening of mitosis or a lengthening -- or 'arrest' -- of interphase. Conversely, increased mitotic index results from a lengthening of mitosis (usually an arrest) or a shortening of interphase. Both of these results were seen among the siRNAs tested. Two of the siRNAs increased the mitotic index more than five fold.

The mitotic arrest phenotypes were then examined by microscopic readout to determine their root causes. This was done by immunofluorescence staining of multiple markers, including tubulin to reveal microtubule distribution. This approach revealed one kinase siRNA that triggered a prometaphase-like arrest state (nearly 16% mitotic index, or ~7-fold higher than normal) wherein spindles were clearly bipolar and chromosomes were well condensed, but with no evidence of successful chromosome alignment (Top Panel, left inset). This likely corresponds to activation of the well-studied spindle assembly checkpoint, which monitors the correct, functional interactions between spindle microtubules and kinetochores in preparation for metaphase and subsequent anaphase onset. A second kinase siRNA that also generated cells in mitotic arrest induced the formation of aberrant spindles displaying either too many or too few apparent poles (e.g. Top Panel, right inset).


Putting It All Together
This screening experiment illustrates the power of higher content, multiparameter assays combined with
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