RNA interference (RNAi) is a powerful tool for studying gene function. By introducing short, double-stranded RNAs (known as small interfering RNAs or siRNAs) that includes the coding sequence of a gene, one can specifically disrupt the function of that gene. In recent years, this technique has become widely used. Currently there are a number of different ways to silence gene expression using RNAi, depending on which model system being studied. In some cases, siRNAs can be chemically synthesized or transcribed
in vitro and then transfected into cells, injected into mice, or introduced into plants (e.g. by a particle gun). In other systems, siRNAs can be expressed endogenously from siRNA expression vectors or PCR products in cells or in transgenic animals.
The Silencer siRNA Construction Kit from Ambion uses in vitro transcription to synthesize large amounts of siRNA. The protocol is quite simple: First design two 29nt oligonucleotides, one for the sense siRNA template and the other for the antisense siRNA template. Within these primers, an 8nt sequence that is complimentary to the T7 RNA polymerase promoter primer must be at the 3’ end of the 21nt target sequence. After the primers have been designed, they are hybridized with the T7 promoter primer (that is included in the kit) and the Klenow fragment is used to fill in the ends for both the sense and anti-sense primers (in separate reactions). Then T7 RNA Polymerase is used to transcribe the siRNA template separately. After each template has been transcribed, the two are incubated overnight at 37oC to generate dsRNA. The next day, an RNase and DNase digestion step will get rid of the DNA template and the overhanging T7 RNA Polymerase promoter sequences. A simple purification step is used to purify the dsRNA (this purification cartridge and reagent are also included with the kit). After purification, normally, I will get about 20 to 40uM siRNA. A detailed protocol can be found online at the Ambion website.
After performing PAGE gel analysis on my siRNA synthesis products, I did see that there were multiple bands in addition to the 21bp band. However, this was most likely the result of having less than completely pure oligos in the initial reactions since using HPLC purified oligos results in a much higher percentage of siRNAs of the right size.
Each kit costs about $500 (US) dollars, which may seem like a lot of money. But it is important to keep in mind that each kit has enough reagents to produce 15 different siRNAs (that is, enough siRNA for 15 different experiments). Thus, this is much cheaper than chemically synthesizing siRNA directly, which can cost $300-400 for each siRNA.
Xiang Yang, Ph.D.
Post-Doctoral Fellow
Georgetown University