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DNA microarrays allow many simultaneous parallel measurements; and transcriptional profiling has provided scientists with a wealth of information. The same technology can be used to build protein arrays which hold similar promise. We describe here an approach using protein arrays to screen cells for gene knockdowns. Small interfering RNAs (siRNA) can be used to degrade mRNA levels of a specific gene, thereby reducing the corresponding protein levels within cells. The use of siRNAs has many implications in functional genomics and proteomics as well as therapeutics, as researchers use this tool to identify proteins involved in specific pathways. We demonstrate the use of microarrays as a screening method to identify cells that have been treated successfully with a beta-actin siRNA.
We have silenced the β-actin gene in HeLa cells using siLentMer Dicer-Substrate siRNA Duplexes, and confirmed the knockdown with a combination of 2-D gel analysis, western blotting and quantitative PCR (qPCR). An increase in the level of phosphorylated cofilin was also detected (Liu, N., et al. 2006 in press).
We now demonstrate the use of reverse phase protein microarrays to screen for these proteins. These arrays enable higher throughput than traditional protein detection, and add multiplexing capabilities. Arrays were produced on the benchtop with the BioOdyssey Calligrapher miniarrayer. Antibodies against β-actin and phosphocofilin were tested for specificity by western blots. In one experiment, arrays were processed to monitor the concentration of β-actin in cells, with a standard curve of purified human actin printed on the grids. In another experiment, changes in phosphorylation levels of cofilin with an antibody specific for phosphocofilin were detected. We also demonstrate that these printed arrays can be screened using antibodies either singly or in pairs. Finally, the microarray results were validated using qPCR.