Small interfering RNA (siRNA), a 20-28 nucleotide double stranded RNA (dsRNA) molecule, also often referred to as “silencing RNA” is used in the gene silencing (or RNA Interference, RNAi) technique to suppress gene expression. The main difference between siRNA and microRNA is that siRNAs are not expressed in humans (or other organisms), while microRNAs are exogenously expressed RNAs that regulate eukaryotic gene activity. Laboratory use of RNAi phenomena using siRNAs to induce gene silencing in mammalian cells provides powerful biology research tool of specific gen function and gene expression, as well as therapeutic potential to cure many diseases. RNAi is a biological mechanism when upon intracellular delivery the siRNA molecules activate RNA-induced Silencing Complex (RISC). Guided by the antisense strand of the siRNA, RISC degrades the target mRNA thus inhibiting its translation.
Gene silencing by RNAi is a powerful research tool for gene expression studies in mammalian cells. This is a biological phenomenon by which small double stranded RNA reduces target gene expression. Inhibition of specific gene expression is experimentally achieved by the transfection of siRNA molecules that binds RISC complex and cause degradation of target complementary mRNA molecules in the cell. Therefore, successful, potent RNAi experimentation is dependent upon the highly efficient delivery of functional (and non-degraded) siRNA into cells. For mammalian cells transfection – the main factor to consider is cell type. There is no transfection reagent that will work well for all cells – both transfection reagent and associated transfection protocol must be optimized for specific tumor cell line or primary cell type. Use of non-optimmized transfection reagents with standard transfection protocol often results in lack of phenotypic response (due to insufficient transfection efficiency), high cytotoxicity, or both.
Most DNA transfection reagents are incompatible with siRNA, sensitive to serum, or are capable of inducing cytotoxicity, which negatively affects gene expression studies. There are commercially available cell line specific transfection kits that have been developed and optimized specifically for use with siRNA transfection and allow transfection in the presence of serum without lowering transfection efficiency or reducing cell viability (see optimized transfection kits). Ready-to-run transfection protocols eliminate the need for extensive siRNA optimization experiments. Altogen Biosystems kits are also compatible for transfection of microRNA (miRNA) molecules.
Neurons siRNA Transfection: Gene Silencing in Neuronal Cells
A number of studies have demonstrated the successful application of RNAi in primary cell cultures and non-neuronal cell lines; however the use of gene silencing in neuronal cell types has been notoriously difficult because of several technical and biological limitations. Neurons have been considered the most resistant to RNAi. Methods to consider when performing nucleic acid delivery experiments with neuronal cultures are: electroporation, microinjection, viral-based methods, and certain chemical transfection reagents. Although physical delivery methods appear to be efficient for siRNA-induced studies where transient modification of gene expression is sufficient, these methods do not appear to be adequate for behavioral studies where long term alteration of gene expression may be necessary. The viral-based siRNA expression systems appear to be most applicable for both Central Nervous System (CNS) gene therapy and basic neurobiological studies, while adeno-associated virus (AAV) delivery systems enable long-term, stable gene expression with relatively little cytotoxicity. Lentiviral vectors have been reportedly used successfully for the delivery of short hairpin RNA (shRNA), a precursor of siRNA, into primary neurons to induce RNAi. The advantage of lentiviral system is the ability to transfect non-dividing cells.
In Vivo Transfection Reagents from Altogen Biosystems
RNAi has been used for in vivo target validation studies using animal models. The major challenge in performing RNAi studies in vivo is the effective, directed delivery of functional small RNA molecules into specific tissues. Altogen® In Vivo Transfection Reagents could be conjugated with siRNA (or microRNA) and administered intratumorally (i.t) or systemically via intravenous (i.v) tail vein injection in order to provide directed gene silencing in specific tissues, including liver, pancreas, kidney, and tumors. Selective knockdown could be seen as early as 24 hours after injection.
Featured in vivo transfection products from Altogen Biosystems:
Altogen Custom Services provide specialized biotechnology and pharmaceutical services, including over 50 validated xenograft models, development of stable cell lines, RNA Interference (RNAi) services, assay development, ELISA and Western Blot services, siRNA library screening and transfection services. Generation of stably-expressing cell lines can be very expensive and time-consuming. Altogen Labs offers generation of stable cell line service completed in just 28 days (see service details).