siRNA Transfection

Summary of siRNA Transfection

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 of 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.

siRNA Design

When designing siRNA, a siRNA library should be used to find any potential off-target effects. Even though a siRNA may match a target sequence, it may also match another sequence in a cell’s genome, and the resulting gene-silencing can result in unpleasant side effects. To some extent, off-target effects will almost always be present, but having sequence specificity and determining the possible off-target effects beforehand can improve experiment results and clarify any unexpected data.

Sometimes, the design of siRNA may be easier with the help of experts in the field. siRNA providers can create the small, hairpin-type inserts utilized for siRNA targets and expression vectors, as well as the provision of analysis and calculations that are targeted or customized to a customer’s specific requirements. siRNA sequence scrambler services provide negative controls for experiments utilizing siRNA; creating scrambled sequences that are identical in nucleotide composition for input sequences, and that pass the same type of filtering processes. The least possible number of matches with mRNA or messenger RNA of particular organisms are also available through siRNA sequence scramblers, offering researchers thousands of combinations and options for study. Such capabilities provide researchers the option of testing and analyzing a number of siRNA processes when it comes to knockdown capabilities, especially for comparison.

A siRNA design service may also provide numerous genomes, in mouse, rat, and human genes. Gene pools (siRNA libraries) offer over 80,000 variations, with such designs saving and maximizing not only time but also optimal experiment results. The availability of such massive numbers of gene pools and their variations allow greater research capabilities and access to experiments than would be available otherwise. When it comes to siRNA design, algorithms can and often include assessment for specifically desired sequences, thermodynamic properties, secondary structures, and so forth.

Optimal siRNA design platforms are made available by the careful documentation of strategies in the design of effective siRNA sequences, ensuring capability of interfacing customized design siRNA for specific gene targeting in a number of fields including life sciences and biomanufacturing. siRNA design companies focus on developing comprehensive services that allow users access to customize siRNA design platforms that offer as much as 80% gene silencing capabilities. Such accessibility is utilized in the study of genomics, proteomics and by the manufacturing products and services.

siRNA Protocols

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.