Using siRNA constructs to selectively knockdown genes of interest has proven to be an essential tool for molecular biologists. Although this technology is in its infancy, it has been quickly accepted as a gold standard for experiments. In addition, libraries of all known gene targets in a studied organism have also been developed, and these siRNA genome libraries have rapidly become the best non-hypothesis driven science to date.
Understanding how all the genes in your organism of interest affect your phenotype can lead to more in depth science and higher impact articles. However, siRNA knockdown does not work with countless tissue culture cell lines and primary cells. Problems have arisen in many primary cells, as well as immune cells, which can be activated by the addition of large amounts of RNA. Dharmacon (now part of Thermo Scientific) has developed a technology that allows previously untransfectable cells to reproducibly achieve significant knockdown. These siRNA constructs with predesigned sequences allow anyone with tissue culture experience the tools necessary for successful knockdown.
After recently learning of its existence, I have used this technology extensively. I primarily work with macrophages, including cell culture lines and primary cells. Our standard transfection protocol using siRNA resulted in efficiencies that were too low for experimentation. In addition, I found that transfection with these high RNA concentrations significantly activated these macrophages. It was difficult to dissect whether the siRNA knockdown or macrophage activation was producing my phenotype. I worked through a couple of transfection protocols, varying reagents, trying electroporation, and eventually giving up on the project.
Serendipitously, I read a paper where the researchers used the new siRNA product from Dharmacon. I obtained the Accell siRNA test kit which contained a fluorescently labeled non-targeting siRNA construct. By flow cyotmetry, I was able to detect over 95 % of my cells fluorescently over two logs above the unstained controls. I was excited, and when the immunoblots showed about a 90 % knock down of my target gene, I was hooked. I have tried about a dozen constructs to date, all with the siRNA smartpools, and I have found a significant knockdown of each of my target genes. However, due to the variables in the siRNA technology, I found a range of knockdown from 70 % to 90 % was common.
Overall, this is a robust new application of the siRNA technology. It does run into one small drawback. Serum in media can significantly inhibit the siRNA delivery process, which is proprietary. Cells must remain in special Accell siRNA delivery media without serum from 48 hours, minimal requirement, to 72 hours, optimal incubation. I suggest purchasing this inexpensive media, and testing the affect of this media on the cells of interest before you commit to the costs of the Accell siRNA.