Roche has analysed the effect of gene knockdown in real time using its Xcelligence system.
The RNA-interference has developed into a powerful tool for loss-of-function studies and has 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, Tschulena et al (Deutsches Krebsforschungszentrum, Division Molecular Genome Analysis, Heidelberg, Germany) analysed 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 60nm concentration, targeting 160 human kinases.
When analysing 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 analyse the effect of gene knockdowns in real time, as the system provides continuous and quantitative information about the electrical impedance at the bottom surface of microtiter plates' wells.
Therefore, 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 less than 0.05.
Moreover, the experiments demonstrate the utility of the Xcelligence system to monitor dynamic effects after knockdown research experiments and the clear superiority of real-time measurements over end-point analysis.
