Analyzing the Effect of Gene Knock-down in Real-Time Using Roche's xCELLigence System
25 Feb 2009The RNA-interference has developed into an extremely powerful tool for loss-of-function studies and has substantially stimulated the application of high-throughput screens. Numerous studies have helped to uncover novel gene functions in many biological processes. Most of these studies have used endpoint analysis as a readout for the respective phenotypes.
In a recent research study (1), Tschulena et al (Deutsches Krebsforschungszentrum, Division Molecular Genome Analysis, Heidelberg, Germany) analyzed the effect of 160 human kinases on cell growth in real-time to monitor the dynamics of the cellular response in response to decreasing levels of the respective kinases. To this end the researchers performed a reverse genetic loss-of-function screen with a small interfering RNA (siRNA) library representing 160 kinases.
To identify kinases having an influence on cell growth, the researchers screened a siRNA library consisting of pools of four siRNAs per gene at 60 nM concentration, targeting 160 human kinases. When analyzing the samples that had been transfected with the siRNA-library, 36 out of the 160 siRNAs did not induce a significant effect when compared to mock or non-targeting control transfected samples. However, knockdown of several genes did induce significant alterations in the growth curve. As expected, knockdown of more than half of the 160 kinases tested induced an inhibitory effect on cell growth. In contrast, the siRNA targeting, for example, EphA4 induced an increase in cellular impedance when compared to non-targeting control transfected samples, indicating an activating effect on cell proliferation.
The experiments demonstrate that the xCELLigence System offers an easy way to analyze the effect of gene knock-downs in real-time, as the system provides continuous and quantitative information about the electrical impedance at the bottom surface of microtiter plates wells. Thus, any change in cell number, cell morphology or cell attachment can be detected in real-time. The xCELLigence System can be well combined with reverse-genetic screening experiments using an automated robotic pipeline such as the Biomek FXP liquid handling workstation. The results show a high reproducibility of the system with the average coefficient of variance being under 0.05. Moreover, the experiments demonstrate the utility of the xCELLigence system to monitor dynamic effects after knock-down research experiments and the clear superiority of real-time measurements over end-point analysis.
The xCELLigence Real-time Cell Analyzer system was originally invented by US-based ACEA Biosciences, is co-developed by Roche and ACEA, and marketed exclusively by Roche. The design of the electronic sensor, innovative techniques to collect and assess data and optimised instrumentation make the system a unique platform for cell-based assays and provide a benchmark potential for cellular and molecular biology. For more information on the technology, please visit follow the company article webpage link on the right hand side of the screen.
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