Small interfering RNA (siRNA) is also known as silencing RNA. siRNA is a double-stranded nucleotide of RNA that performs a function in a biological system. A siRNA transfection is the “transference” of genetic material into a cell and is involved in gene silencing experiments. In order to optimize a siRNA transfection, the correct method and transfection agent will lead to increased efficiency and desired results. The optimization of siRNA transfection depends greatly on the cell line used, as well as siRNA transfection protocols.
RNAi response has been effectively demonstrated in mammalian tissues and cells for exogenous and endogenous genes. The gene knockdown effect can be partial silencing of gene expression or complete, thus turning off the function of an entire gene. Long dsRNA (>30 base pairs) is known to trigger an interferon response which leads to general mRNA cleavage and apoptosis. Thus, length of the designed siRNA is one of the crucial parameters to control in the manufacturing of RNAi products. http://www.ncbi.nlm.nih.gov/genome/probe/doc/TechRnai.shtml
Scientists have discovered that small interfering RNA (siRNA) are extremely valuable in silencing gene expression and enables studying gene functions in a multitude of cells. The success of RNAi experiments relies on method of delivery of siRNA or miRNA. siRNA can be transiently or stably transfected using transfection reagents. In many cases, cell types such as primary cells may make lipid based transfections difficult, limited or even impossible.
Determining optimal transfection parameters can result in the success or failure of RNAi effects in various cell cultures. Parameters to optimize includes the type and volume of transfection agent used, culture conditions, the exposure time transfection agents are on cells, as well as the quality, purity and quantity of siRNA used in the experiments.
Choosing the correct procedure to follow is also critical. For example, a forward transfection procedure enables cells to attach and recover from the cell collection process and grow for 24 hours prior to transfection. In some situations, a reverse transfection may offer enhanced benefits over the more traditional forward methodology.
Other considerations for a successful siRNA transfection include the current health of the cultured cells, the conditions under which transfection occurs and the method of transfection that is followed. Cells must be extremely healthy in order to ensure maximum viability. Healthy cells are easier to transfect than damaged, poor quality cells. Various methods and adequate number of cells will help ensure the health and success in experiments. Also, cell density, transfection reaction volume and exposure time of reagent on the cells plays an important role in the success or failure of siRNA transfection experiments.