Small interfering RNA (siRNA), also known as silencing RNA, is a double-stranded segment of RNA that can perform various functions in a biological system. A siRNA transfection is the insertion of siRNA into a cell, a process that can be invaluable to gene silencing experiments. In order to optimize a siRNA transfection, the correct method and transfection agent should be used. The optimization of siRNA transfection depends greatly on the cell line used, which may require the tailoring of transfection reagents to a specific cell type.
RNAi response has been effectively demonstrated in mammalian tissues and cells for exogenous and endogenous genes. The gene knockdown effect can result in partial silencing of gene expression or the complete elimination of 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, the length of the designed siRNA is one of the crucial parameters to control in the manufacturing of RNAi products.
Research has shown that small interfering RNA (siRNA) is extremely valuable in silencing gene expression and enables the studying gene functions in a multitude of cells. The success of RNAi experiments relies on the 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, requiring the use of more potent transfection techniques.
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, exposure time, 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.