RNAi as a Tool
The use of RNAi is fast becoming an important tool to silence gene expression in order to modulate cellular function. To achieve optimal silencing, several factors must be considered, according to Kathy Latham, Ph.D., senior product manager for Ambion (www.ambion.com), an Applied Biosystems business. “The design of siRNA is important. The trick is to find one that efficiently degrades the specific mRNA of interest. We have formulated sophisticated siRNA design and specificity checking algorithms to help with this. These take into account such things as melting temperature and nucleotide locations. Experimentally, the siRNA concentration used is also critical. One should use the least amount in order to minimize off-target effects.”
In addition to siRNA tools, Applied Biosystems also focuses on another player in RNA-directed gene expression, miRNA. The genomes of plants and animals are chock full of these small single-stranded RNAs that impact a variety of important biological processes.
“MicroRNA are naturally occurring regulatory molecules that primarily act at the level of translation. They are believed to regulate 50% or more of all proteins and play critical roles in a variety of different processes from fat metabolism to cancer,” Dr. Latham explains. It is unusual to find a pathway not impacted. But one needs specialized methods to study miRNAs.”
Applied Biosystems provides a host of tools for studying miRNAs. “Our TaqMan® MicroRNA Assays allow one to amplify specific small RNAs to accurately quantify microRNA expression levels and characterize microRNA expression via real-time RT-PCR,” Dr. Latham says.
Aside from studying the expression patterns of miRNAs, one really needs to define the target(s) of the miRNA, explains Dr. Latham. “Bioinformatics can help, but the state-of-the-art right now is imperfect at best. To really understand function, one needs to inhibit or mimic microRNA function. We have developed Pre-miR™ miRNA Precursors that mimic endogenous microRNAs to determine microRNA’s biological effects via gain-of-function experiments. This helps to better understand their impact on the protein you think is being regulated.”
A new methodology developed by Mirus Bio (www.mirusbio.com) involving a simple intravenous injection of large fluid volumes is helping researchers deliver genes or siRNA molecules more efficiently to liver hepatocytes. Richard S. Schifreen, Ph.D., vp of research products, says, “It’s long been desired to have a means to effectively introduce genes into the liver. Our protocol, referred to as hydrodynamic intravascular injection, involves the high-pressure, rapid delivery of sample into the vein using enough volume of nucleic acid solution (naked DNA) to target cells outside of the blood vessel.
“For RNA interference applications in our rodent models, we routinely inject siRNAs into the tail vein and this allows generation of knock-down adult animal models in days without impacting embryonic development.”
Dr. Schifreen says that the inherently simple method also results in low toxicity. “This procedure is quick, only takes a few minutes to perform, and we see 10–40% transfection efficiency into hepatocytes. This is a great improvement over traditional delivery methods that use nonviral DNA or siRNA methodologies. These often only achieve 0–1% efficiency. It is also an exquisite tool in that you can perform both knockdown and then rescue in the same animal. This can help the researcher determine how a gene affects the phenotype in the pathway being studied.”
Mirus Bio is also focusing on the analysis of miRNA expression using microarray analyses. “We have developed a single-step alkylating system to bind the tag to nucleic acids, which allows us to track the nucleic acid throughout various systems, tissues, or even cells. We are applying this to studying microRNA. This labeling system, called Label IT, is sequence independent and does not change the sequence of the labeled microRNA. This is important because microRNAs are small and other commercially available labeling reagents require attachment of additional sequences that result in a failure to detect all of the microRNA species present in the sample.”