One way in which to study the functions of a particular gene is to prevent the expression of that gene. Silencing gene expression will eliminate the mRNA available for protein translation. Once a particular protein is no longer translated, key functions mediated by the protein within the cell or the organism will be lost or disturbed, and the researcher can then begin to deduce its critical roles. Recent discoveries have shown that double-stranded RNA (dsRNA) can silence the expression of a gene that is homologous to one of the strands within the duplex. This phenomenon, termed RNA interference (RNAi), was first described in plants, and was also shown to be highly effective in suppressing gene expression in higher organisms such as Drosophila melanogaster and the nematode Caenorhabditis elegans. More recently, gene silencing by dsRNA has been demonstrated to be successful in mammalian cell culture, thus facilitating the studies of specific genes and protein functions in mammals. Researchers interested in employing RNAi techniques in mammalian cell culture in their laboratories should consider synthesizing their siRNA oligonucleotides at Dharmacon Research, Inc. (Lafayette, CO). The company has extensive experience with dsRNA synthesis and can be found online at www.dharmacon.com.
Although the mechanisms for RNAi remain unknown, the steps required to generate the specific dsRNA oligonucleotides are clear. It has been shown that dsRNA duplex strands that are 21-26 nucleotides in length work most effectively in producing RNA interference. Selecting the right homologous region within the gene is also important. Factors such as the distance from start codon, the G/C content and the location of adenosine dimers are important when considering the generation of dsRNA for RNAi. Fortunately, specific and useful details can be found on the Dharmacon website. One consequence of this, however, is that one may need to test several different sequences for the most efficient RNAi and this may become costly. The oligonucleotides arrive in several different aliquots, and after a key step, in which stabilizing 2’ protection groups are removed, the dsRNA is ready for incorporation into cells by transfection.
The pricing for oligonucleotide synthesis at Dharmacon is competitive and there is a quick turn-around time between placing the order online and receiving the products (about one week). And we have found that the Customer Service representatives are also very helpful should one have technical questions. Overall, we have had a good experience buying our dsRNA oligonucleotides from Dharmacon Research.
Joe F. Lau
Graduate Student
Mount Sinai School of Medicine, NY.