Gene Silencing Solutions with the Agilent 2100 bioanalyzer
Gene silencing with RNA interference (RNAi) is a new valuable technology for functional genomics in target validation during drug discovery. Scientists working with gene silencing require effective and easy-to-use tools to optimize their experiments.
 Optimization of siRNA transfection procedure
siRNA delivery into cells is critical in any gene silencing experiment. Optimization of procedure is required when different cell lines or transfection methods are used. Optimize transfection conditions for gene silencing experiments with the help of fluorescently labeled siRNA and cell fluorescence assay of the Agilent 2100 bioanalyzer. Cell viability and delivery efficacy are simultaneously evaluated with fluorescent probes, therefore helping to decide on the optimal transfection conditions. Little amounts of fluorescently labeled siRNA are required for the optimization. The flow cytometry measurements needs only 20,000 cells/sample and the transfection is independent on the siRNA sequence, therefore one fluorescently labeled siRNA can be used for many siRNA optimization experiments.
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 Correlation of siRNA uptake and gene knockdown
A simple co-transfection experiment of a plasmid expressing GFP protein and a red-fluorescent labeled siRNA against GFP can verify RNAi mechanisms in the cell line of choice.
Cells in panel A were transfected only with red-fluorescent labeled siRNA and a blue live cell dye Calcein. The Agilent 2100 bioanalyzer measured a high transfection efficiency as 88.7% of all live cells were positive for Cy5-siRNA. Panel B shows result of cells that were transfected only with a plasmid coding for GFP. Here 51% of all live cells express the GFP protein. Panel C demonstrates that the silencing mechanism is working. These cells were transfected with both, GFP plasmid and siRNA against GFP, which results in a silenced GFP expression with 3% of all cells after 24 hours.
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 Verify RNAi silencing by cellular protein expression measurement
The goal of gene silencing is to deactivate a selected target. Ideally, this is verified by a decrease in target protein expression. A decrease of respective mRNA might also be a good indication for successful gene silencing. However, measurement of protein expression on a cellular level takes into account that different proteins have different life times. Experimental data shows silencing of the nuclear protein lamin, which is known to have a long half life. Already 24 hours after transfection a significant silencing effect was measureable with the flow cytometry capability of the Agilent 2100 bioanalyzer by intracellular antibody staining. Once protein expression has been proved, additional measurements for functional analysis can be performed.
 Detection of siRNA
Small interfering RNA is double stranded and can be quickly analyzed with the DNA 1000 Labchip kit of the Agilent 2100 bioanalyzer. Checking siRNA for impurities, digestion and degradation before its usage in expensive cell experiments is recommended.
Related topics
Gene expression solutions
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