RNA Interference in Mammalian Cells Using low siRNA Concentrations
Poster Feb 01, 2007
Jörg Dennig, Silvia Magyar, Anja Grewe, Cornelia Schmidt, Peter Hahn, Dong Liang, Subu Yerramilli, Eric Lader, Wolfgang Bielke, and Jie Kang
The use of short interfering RNA (siRNA) for knockdown of gene expression has become a powerful tool in molecular and cell biology. Some applications require the use of low siRNA concentrations (less than 5 nM), for example, to decrease the possibility of non-specific effects.
We have developed a transfection reagent, HiPerFect Transfection Reagent, which allows efficient gene knockdown with siRNA concentrations from 1 nM–10 nM, depending on the cell type and siRNA used. HiPerFect Transfection Reagent has been tested and validated for many cell types, including primary cells. Effective knockdown in primary cells demonstrates that HiPerFect Transfection Reagent ensures low cytotoxicity levels.
A Fast-Forward siRNA Transfection Protocol has been developed for rapid transfection with HiPerFect Transfection Reagent. This protocol allows cell seeding and transfection on the same day.
A reverse transfection protocol has been developed that is ideal for use in high-throughput applications. In reverse transfection, siRNA is spotted into wells, followed by addition of HiPerFect Reagent. After complex formation, cells are added to the wells.
When there is a need to quickly analyze samples using a number of different PCR assays, it is likely that optimal conditions for each assay will not be the same. First, different assays often will require different annealing temperatures for their primers. In addition, amplicons may be designed to be of different lengths and therefore require varying durations of the extension step.READ MORE