Analyzing the Effect of Gene Knock-down in Real-Time Using Roche's xCELLigence System
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 www.xcelligence.roche.com.
(1) Jitao David Zhang, Angelika Duda, Christian Schmidt, Anja Irsigler, Stefan Wiemann, Ulrich Tschulena, Biochemica (2009).
About Roche Diagnostics Deutschland GmbH
Headquartered in Basel, Switzerland, Roche is one of the world's leading research-focused healthcare groups in the fields of pharmaceuticals and diagnostics. As the world's biggest biotech company and an innovator of products and services for the early detection, prevention, diagnosis and treatment of diseases, the Group contributes on a broad range of fronts to improving people's health and quality of life. Roche is the world leader in in-vitro diagnostics and drugs for cancer and transplantation, and is a market leader in virology. It is also active in other major therapeutic areas such as autoimmune diseases, inflammatory and metabolic disorders and diseases of the central nervous system. In 2008 sales by the Pharmaceuticals Division totalled 36.0 billion Swiss francs, and the Diagnostics Division posted sales of 9.7 billion francs. Roche has R&D agreements and strategic alliances with numerous partners, including majority ownership interests in Genentech and Chugai, and invested nearly 9 billion Swiss francs in R&D in 2008. Worldwide, the Group employs about 80,000 people. Additional information is available on the Internet at www.roche.com.
(1) Höper D, Hoffmann B, Beer M. Simple, sensitive, and swift sequencing of complete avian influenza H5N1 genomes. (2009) Journal of Clinical Microbiology.
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