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Confirming gene silencing,,,mechanism by pGFP/GFP22 ,,,siRNA co-transfection

In addition to the specificity and efficiency of an siRNA sequence for a target gene, which are of high importance in post-transcriptional gene silencing (PTGS), the delivery of siRNA into cells is crucial. An efficient method to optimize transfection and gene silencing is critical for successful RNAi experiments. In this Application Note HeLaS3 cells were transfected with GFP expressing plasmid and Cy5-labeled siRNA against GFP, both individually and in combination, using QIAGEN transfection reagents. Uptake of labeled siRNA and the gene silencing effect were monitored for up to 24 hours after transfection. Efficient gene silencing was achieved which confirms the operation of RNAi mechanism in the selected cell line.
The technique of RNAi has the potential to revolutionize functional genomics. The pathways and mechanisms of RNAi are currently under intense investigation, and a good understanding of these is essential to allow scientists to exploit the applications of RNAi. Simple and efficient methods for monitoring gene silencing are important tools for RNAi experiments. In this Application Note we describe the evaluation of a method using fluorescent labels to determine transfection efficiency and the gene silencing effect.
HeLaS3 cells were transfected with siRNA using RNAiFect Reagent. TransMessenger Reagent was used for the transfection of GFP expressing plasmid (pGFP). Cells were also co-transfected with both plasmid DNA and siRNA using TransMessenger Reagent. The uptake of siRNA into mammalian cells was monitored using fluorescent labeled siRNA and the gene silencing effect and level of cellular protein expression were quantified at 4, 8, and 24 hours after transfection.

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1. Confirming RNAi Effects Is Now Easier Than Ever
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