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siRNA transfection optimization,,,with the Agilent 2100 bioanalyzer

When working in the field of target validation, or when studying functional assays requiring the transfection of small interfering RNA (siRNA) into a cell, the presence of having a fast, easy-to-use method to optimize transfection conditions for given cell lines would be of great value. Three to four siRNA sequences are generally tested for each gene in order to suppress the expression of a protein with a corresponding mRNA sequence. Before any sequence comparison can be performed, the integrity and purity of siRNA, the siRNA uptake and the cell viability must be monitored and optimized. Once optimal transfection conditions have been established, different siRNA sequences can be evaluated for the lower target protein expression. There are many influencing factors for transfection efficiency, among them the cell line, siRNA concentration and its ratio to the transfection reagent, cell confluence during transfection, incubation time and media composition all need to be taken into consideration. In this Application Note we describe the use of Agilents 2100 bioanalyzer to quickly verify the siRNA integrity and to determine the optimal transfection conditions for gene silencing experiments with the help of red-fluorescently labeled siRNA and cell fluorescence assays. Cell viability and delivery efficacy are simultaneously evaluated with fluorescent probes, therefore helping to decide on the optimal transfection conditions.
Over recent years the scientific community has seen an explosion in the number of research studies related to RNA interference (RNAi) induced gene silencing. Benefits, such as genome usability to generate knockout phenotypes, easy automation with the potential for high throughput, specificity, and low cost when compared to animal knockout models are seen
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