RNA interference (RNAi) is a powerful technique for down regulating the expression of specific genes in live cells. In mammalian cells, RNAi can be induced by transfecting small interfering RNAs (siRNAs), comprising double-stranded RNA molecules ~21 nt in length with 2 nt 3' overhangs. In order to find an siRNA with a strong effect, 35 siRNAs per gene are typically designed. This means synthesizing and purifying 610 RNA oligonucleotides. The Silencer siRNA Construction Kit produces transfection-ready siRNA at a fraction of the cost of chemical synthesis. The cost and time savings gained using the Silencer siRNA Construction Kit enable screening of more genes and more potential targets within your gene to find the most potent siRNA. Higher potency siRNA translates to better signal-to-noise ratios in your experiments and fewer nonspecific effects.
How the Kit Works
Construction of an siRNA begins with the acquisition of two inexpensive, desalted DNA oligonucleotides. As illustrated in Figure 1, these oligonucletides include an 8 base sequence complementary to the 3' end of the T7 promoter primer include d in the Silencer siRNA Construction Kit. Each gene specific oligonucleotide is annealed to the supplied T7 promoter primer. A fill-in reaction with Klenow fragment generates a double-stranded template ready for use in an in vitro transcription reaction. The two transcription reaction products are hybridized to each other, treated with DNase (to remove template) and RNase (to polish the ends of the double-stranded RNA), and column purified. The entire procedure can be completed in less than 24 hours and is easily scalable -- 15 siRNA molecules can be synthesized as easily as a single siRNA. Each transcription/purification produces enough siRNA for hundreds of transfections. The time required for double-stranded RNA synthesis and purification using the Silencer siRNA Construction Kit is a fraction of the 12 week processing time typically required for RNA oligonucleotide synthesis via phosphoramidite chemistry and subsequent purification.
siRNAs prepared using the Silencer siRNA Construction Kit were used to silence GFP expression in HeLa cells (Figure 2).
Figure 1. The Silencer siRNA Construction Kit Procedure .
Figure 2. siRNAs Prepared with the Silencer siRNA Construction Kit Efficiently Induce RNAi. Four siRNAs to different target sequences of GFP were synthesized and purified with the Silencer siRNA Construction Kit. The siRNAs were co-transfected with a plasmid expressing GFP into HeLa cells. Cells were analyzed 24 hours after tra nsfection by fluorescence microscopy. (A) GFP expression plasmid only. (B) GFP expression plasmid co-transfected with siRNA #2. (C) Silencing efficiency achieved with the 4 different siRNAs. Note the variation of siRNA potencies with different target sites.
The kit includes all necessary reagents to generate 15 purified double-stranded siRNAs ready for transfection. Also included is a detailed Instruction Manual and GAPDH control templates. The control templates can be used to verify that the kit is functional, and the GAPDH-specific siRNA made in the control reaction can be used for optimizing transfection protocols.