Sidebar: Assessing Gene Function in Real Time
One of the challenges in RNAi research is choosing an appropriate robust cell-based functional assay for obtaining important information about the phenotypic effect of targeted gene knockdown, discriminating off-target and toxic effects. Both off-target interactions and toxicity are an inherent part of the RNAi approach, dependent on the RNAi target sequence, concentration, and cell type used.
In a new application note (“Using the xCELLigence™ System for Functional Genomics: Assessing Gene Function in Real Time”), investigators from Roche Applied Science describe use of the instrument, which allows for label-free continuous monitoring of changes in cell phenotype using microelectrodes to record electrical impedance, for assessing RNAi-mediated knockdown of gene function.
Cells were seeded in standard 96-well microplates, manufactured with integrated microelectronic sensor arrays called E-Plates 96. The interaction of cells with the electronic biosensors generates cell-to-electrode impedance responses, measuring many aspects of cell status, including cell number, cell viability, cell morphology, and the quality of cell attachment. Real-time, continuous measurement results in uninterrupted documentation of cell phenotype by producing time-dependent cell response profiles (TCRPs), according to the Roche team, who add that TCRPs provide predictive information about how cells and cellular pathways are responding in vitro.
Kinetic information revealed by TCRPs can discriminate siRNA-mediated off-target and toxic effects. The focus in this application note was on several genes implicated in the mitotic pathway.
HeLa and A549 cells, obtained from ATCC, were maintained in DMEM media with 10% FBS and 1% penicillin and streptomycin, at +37ºC with 5% CO2. Cell attachment, spreading, and proliferation were continuously monitored every 30 minutes on E-Plates 96 using the xCELLigence system.
The electronic readout of cell-sensor impedance is displayed continuously in real-time as the cell index (CI). The CI value at each time point is defined as Rn-Rb/Rb, where Rn is the cell-electrode impedance of the well with the cells, and Rb is the background impedance of the well with media alone.
Assessing Gene Function after Knockdown
To better document the correlation between target gene expression, CI changes, and mitotic arrest, threefold serial diluted KIF11 siRNA (KIF11-7904) was transfected into HeLa cells in E-Plates 96 for cell index measurements, and 16-well chamber slides for quantifying mitotic index; 24 hours post transfection, cells were harvested from E-Plates 96 to quantify KIF11 mRNA levels, or fixed and stained using p-H3 antibody in chamber slides to determine mitotic indices. TCRPs showed time- and concentration-dependent dose response curves, with higher siRNA concentrations producing more CI changes.
The kinetics of CI changes were very similar for the different concentrations of siRNAs. CI values for transfected samples diverged from control samples, starting 9–12 hours post transfection, reaching the lowest level at 24 hours post transfection, before the CI started recovering. CI changes were highly correlated to target mRNA levels and mitotic indices, with higher siRNA concentrations producing more pronounced CI changes, resulting in higher target downregulation and a higher mitotic index.
Interestingly, the Roche scientists note that siRNA transfected at a concentration of 0.01 nM still produced robust CI changes and target downregulation. Similar dose responses were also observed for PLK1-448 and PLK-450 siRNAs, while no dose response was seen for control siRNAs.
The dose-dependent TCRPs obtained allow for the determination of the efficacy of target knockdown on a particular phenotype, explain the Roche researchers.