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Five Ways to Produce siRNAs

l Kit (RNase III)
With the Silencer siRNA Cocktail Kit (RNase III), a population of siRNAs to a specific target can be prepared in a quick, simple procedure. The population of siRNAs produced by the Silencer siRNA Cocktail Kit (RNase III) elicits gene silencing effects comparable to well designed individual siRNAs, without the need to design and screen individual siRNAs. Ambion scientists have tested many genes and they have yet to see an increase in cytotoxicity. They have also not observed any nonspecific effects associated with the use of siRNA populations as compared to individual, chemically synthesized siRNAs targeting the same gene. To date, Ambion scientists have used this kit successfully with NIH-3T3, HeLa, S3, 293 and BJ cell lines and have knocked down the expression of numerous genes, including c-fos, GAPDH, La, -actin, and Ku-70.

Figure 2. Gene Silencing with the Silencer siRNA Cocktail Kit. A population of siRNAs targeting 200 nt of the Ku-70 mRNA was prepared with the Silencer siRNA Cocktail Kit (RNase III) and transfected into HeLa cells at a final concentration of 100 nM. Cells were analyzed 48 hours later by immunofluorescence. Ku-70 levels were reduced 86% in cells transfected with the siRNA cocktail, compared to non-transfected controls.


In Vivo Expression

All of the methods described so far rely on the in vitro preparation of siRNAs. The use of siRNA expression vectors and PCR-based expression cassettes, h
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