A new Approach to Functional Screening of siRNA Knockdown
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Guava Technologies, Inc. presented at the recent Molecular Targets & Cancer Therapeutics Symposium information on their recent advancements that describe an experimental methodology and the new Guava® Simplicity Analysis Software which exploits the advantages of plate based flow technology.
These technological improvements result in an overall process that can expedite the drug discovery process by providing a means for extraction of key findings from the highly complex data sets encountered with functional screening of siRNA knockdown assays.
Solid tumors comprise genetically heterogeneous cell populations whose growth and survival depends on the complex interplay of distinct, yet overlapping, signaling networks. A major challenge in developing a course of therapy is determining which signaling nodes to target for a specific malignancy.
Profiles from siRNA gene silencing are integral to mapping disease-specific signaling cascade(s) and provide insight to key targets for therapeutic intervention. Successful siRNA screening relies not solely upon optimizing transfection, but also cell analysis systems capable of high content screening at the single cell level, within overall populations (sample well), and across multiple data sets.
The presentation describes how the Guava EasyCyte™ Plus System, with integrated Guava Simplicity Software, provides a revolutionary platform for secondary target validation and compound screening.
Guava Technologies' flow cytometers overcome the limitations of inference-based measurements of transfection efficiency and protein knockdown through direct quantitiative analysis of populations at the single cell level.
The Simplicity Analysis Software's intuitive architecture and facilitates the process of asking biological questions on multi-dimensional data sets through visualization of user-defined parameters in the form of heat-maps. Most importantly, comparative results are displayed at the experiment level rather than on an individual well/sample basis.
Specifically, using the EasyCyte Plus System in tandem with Simplicity Analysis Software, 23 agents were identified that had growth restrictive properties although significant variation across cell lines was observed.
Further targeted gene knockdown via siRNA confirmed the presence of both activators and inhibitors of Camptothecin-induced apoptosis as well as gene targets for growth arrest.
Screens for apoptosis and cell cycle, as well as phospho-signaling intermediates, defined compounds with mechanisms of action similar to and different from Camptothecin. Cell-based assays for phenotype and function revealed a number of cooperative and antagonistic interactions between signaling intermediates, their respective cascades, and cytoactive agents.
Overall, the acquired multiplex data set is shown to provide a more detailed view on the behaviour of each of the test compounds with respect to apoptotic induction, cell cycle progression, and the signaling cascades that regulate these cellular responses to drug treatment.
In total, this experimental methodology, when used in conjunction with Guava Technologies' cell analysis platforms and Simplicity Analysis Software, expedites the drug discovery process by providing a means for extraction of key biological findings from complex result sets.
