RNA interference (RNAi) is a well-established technology that revolutionized the way that researchers study mammalian gene expression and continues to contribute valuable insights into gene function today. RNAi has had significant impact on the ease, speed, and specificity with which the loss of gene function analysis can be done in mammalian cells and animal models and has long been a method of choice for researchers doing loss of function studies.
Gene over expression has been used to analyze the function of genes and their role in disease. However, the phenotypes resulting from over expression may not reflect the actual gene function. Dominant negative constructs have been used; however this will not work for every protein, and results can be hard to interpret. Knockout mice can be developed to understand protein function. This method is laborious and expensive and may result in an embryonic lethal phenotype. Antisense and ribozymes have been used, yet these may not work for all targets, and RNAi tends to be more potent than these other methods.
The human genome contains thousands of target genes that are candidates as druggable targets. RNAi technology can be effectively utilized in target validation to identify and functionally assess relevant disease genes. RNAi technology can also be used as a tool to evaluate gene expression signatures in response to lead compounds or signaling pathways. Ultimately, RNAi will be a powerful tool in animal models with applications being developed to use stable modified molecules such as Stealth™ RNAi or lentiviral delivery for effective in vivo studies.
Figure 1. Evolution of RNAi
RNA Interference (RNAi) is one of the most important technological breakthroughs in modern biology, allowing us to directly observe the effects of the loss of function of specific genes in mammalian systems.
In the early 1990s, a number of scientists observed independently that RNA inhibited protein expression in plants and fungi (Figure 1). This phenomenon, identified but not understood, was then known as “posttranscriptional gene silencing” and “quelling”. In 1998 Fire and Mello observed in Caenorhabditis elegans that double-stranded RNA (dsRNA) was the source of sequence-specific inhibition of protein expression, which they called “RNA interference”. While the studies in C. elegans were encouraging at that time the use of RNAi as a tool was limited to lower organisms because delivering long dsRNA for RNAi was nonspecifically inhibitory in mammalian cells. Fire and Mello won the 2006 Nobel Prize in Physiology or Medicine for their discovery of RNA interference.
Further studies in plants and invertebrates demonstrated that the actual molecules that led to RNAi were short dsRNA oligonucleotides, 21 nucleotides in length, processed internally by an enzyme called “Dicer”. The Dicer cleavage products were referred to as “short interfering RNA” now popularly known as “siRNA”. Subsequently in 2001, it was demonstrated that siRNA could directly trigger RNAi in mammalian cells without evoking nonspecific effects.
Today we have a greater understanding of the components that are part of the RNAi pathway, the efficiency with which these components function, the specificity of sequence recognition and cleavage of cellular mRNA and many of the key requirements for designing and generating extremely effective RNAi reagents. Our analysis of novel chemistries will further improve this approach and lead the way to in vivo analysis and potentially make the use of RNAi in therapeutics possible.
RNAi technology can be used to identify and functionally assess the thousands of genes within the genome that potentially participate in disease phenotypes. In addition, RNAi technology provides an efficient means for blocking expression of a specific gene and evaluating its response to chemical compounds or changes in signaling pathways.
RNAi technology takes advantage of the cell’s natural machinery, facilitated by short interfering RNA molecules, to effectively knock down expression of a gene of interest. There are several ways to induce RNAi, synthetic molecules, RNAi vectors, and in vitro dicing (Figure 2). In mammalian cells, short pieces of dsRNA, short interfering RNA (siRNA), initiate the specific degradation of a targeted cellular mRNA.
In this process the antisense strand of the siRNA duplex becomes part of a multi-protein complex, or RNA-induced silencing complex (RISC), which then identifies the corresponding mRNA and cleaves it at a specific site. Next, this cleaved message is targeted for degradation, which ultimately results in the loss of protein expression.
Figure 2. Methods of RNAi knockdown in mammalian cells.
Our Technical Application Scientists are available to help assist you at email@example.com
Ordering & Order Status inquires:
If you have questions about pre-designed RNAi orders and order status, please contact us at firstname.lastname@example.org
If you have any questions about Custom RNAi orders and order status, please contact us at RNAiSupport@thermofisher.com
For Research Use Only. Not for use in diagnostic procedures.