( to inhibit cell proliferation arrest ...)siRNA screening of the cell cycle with two dynamic GFP sensors (from Discovery Matters Issue 1, July 2005)

to inhibit cell proliferation, arrest cells in mitosis, and induce apoptosis (15). Cell cycle analysis (Fig 6, PLK) of G2/M CCPM cells treated with siRNA directed against PLK showed a dramatic increase in mitotic cells 48 h after transfection with 50 nM siRNA (Fig 8B). Extreme sensitivity to PLK knockdown was confirmed by analysis of G1/S CCPM (Fig 8A) and G2/M CCPM (data not shown) 24 h following transfection with 5 nM siRNA, which showed an increase in G2 and M phase cells and a corresponding decrease in G1 cells.

One key advantage of high content cellular analysis is the ability to analyze high resolution images for multiple parameters. In this case additional morphological analysis of images derived from cells treated with Cyclin A2 siRNA revealed a significant increase in nuclear area (395.3 ± 173.7 µm²) compared with control cells (219.1 ± 95.7 µm²) and Cyclin A1 siRNA treated cells (229.8 ± 98.5 µm²).

Re-analysis of all image data from the siRNA library screen using DNA granularity revealed that PLK siRNA gave the most significant induction of apoptosis across the target genes in this study (Fig 8C).

Conclusions
Perturbation of sensitive and dynamic phenotypic cellular assays via siRNA provides a powerful tool for functional analysis of the cell cycle. High-throughput sub-cellular imaging and automated multi-parameter image analysis provides an information rich environment to screen and study effects of gene knockdown with siRNA.

References
1. Hannon, G.J. RNA interference. Nature 418 (6894), 244–251 (2002).

2. Paddison, P.J. and Hannon G.J. siRNAs and shRNAs: skeleton keys to the human genome. Curr. Opin. Mol. Ther. 5 (3), 217–224 (2003).

3. M
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