No more siRNA design and testing
Entire procedure complete in 1 day
Includes reagents for long dsRNA preparation, RNase III digestion and siRNA cleanup
Simplify Your Gene Silencing Experiments
The use of small interfering RNAs (siRNAs) to induce targeted gene silencing in mammalian cells is now a common method for analyzing gene function. One drawback to the technology, however, is the need to design, synthesize, and test several siRNAs before an effective sequence is identified. This involves considerable time and expense. Now the "siRNA design problem" can be avoided altogether. With the Silencer siRNA Cocktail Kit (RNase III), researchers can prepare a population of siRNAs to a specific target in a quick, simple procedure. This population of siRNAs elicits gene silencing comparable to validated individual siRNAs without the need to design and test individual siRNA sequences.
Preparing siRNA Cocktails
Recently it has been shown that a population of siRNAs generated by digesting long dsRNA with RNase III can be used to effectively induce RNAi in mammalian systems (14). RNase III cleaves dsRNA into 1230 bp dsRNA fragments with 2 to 3 nucleotide 3' overhangs, and 5'-phosphate and 3'-hydroxyl termini. The termini and overhangs of RNase III cleavage products are thus the same as those produced by Dicer in the in vivo RNAi pathway.
The population of siRNAs produced by the Silencer siRNA Cocktail Kit (RNase III) elicits gene silencing effects comparable to well designed specific siRNAs (Figure 2). Ambion scientists have tested many genes and they have yet to see an increase in cytotoxicity nor have they seen any nonspecific effects associated with the use of siRNA populations as compared to individual chemically synthesized siRNAs targeting the same gene (3).
Complete Kit for siRNA Cocktail Production
The Silencer siRNA Cocktail Kit (RNase III) includes reagents to synthesize long dsRNAs by in vitro transcription with T7 RNA polymerase, and to digest these dsRNAs into siRNA cocktails using RNase III. Reagents are provided to prepare siRNA cocktails to 20 different genes, with each RNase III reaction and subsequent purification producing enough siRNA for over a hundred transfections in 24 well plates. siRNA Purification Units are also available separately.
Figure 1.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.
Figure 2. siRNA Cocktails Are as Effective as Individual siRNAs A population of siRNAs targeting the first 200 bases downstream of the AUG start site of GAPDH mRNA was prepared with the Silencer siRNA Cocktail Kit (RNase III). The resulting siRNA cocktail, or a chemically synthesized siRNA known to efficiently silence GAPDH, was transfected into HeLa cells. GAPDH protein levels were analyzed by immunofluorescence 48 hrs later.