Long-term Gene Silencing in Mammalian Cells using siRNA
Poster Feb 01, 2007
Frank Narz, Silke Janhsen and Martin Weber
Gene silencing using siRNA-mediated RNAi has become a powerful tool in cell biology, addressing a range of research questions in functional genomics and drug discovery. After delivery of siRNA into the cell, rapid degradation of the target mRNA can be detected, often within 24 hours, and subsequently the corresponding protein is also knocked down.
This knockdown is transient, especially when working with proliferating cell lines where the intracellular siRNA is diluted with each cell division. As most cell lines have to be subcultured approximately twice a week, siRNA-mediated RNAi experiments are usually analyzed 3–4 days after transfection. In many cases, this short-term knockdown is too brief and phenotypic effects that require a longer duration of knockdown of the target protein cannot be examined.
We have developed a protocol that allows gene silencing in mammalian cells for more than 2 weeks. siRNA is transfected using lipid-based HiPerFect Transfection Reagent every time the cells are diluted and plated into a new culture plate.
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