Until recently, gene knockdown or knockout technologies, such as antisense, ribozyme, and gene deletion, were used to perform loss-of-function studies. However, the post-genomics era called for high-throughput gene function studies for which the above technologies were unable to answer due to poor reproducibility, high cost, and labor-intensiveness.
The advent of siRNA technology has opened up many new possibilities in the field of gene suppression. The term siRNA is generally used for 21-nucleotides RNA duplexes, where the two terminal 3'-nucleotides are unpaired (3' overhang). The siRNAs are introduced into cells where they are unwound and form complexes with RISC. The siRNA strand complementary to the target mRNA is incorporated into the RISC. The RISC complex then recognizes and cleaves the target mRNA, which leads to the degradation and silencing of the target mRNA.
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RNA interference (RNAi)
RNA interference (RNAi) or gene silencing involves the use of double stranded RNA (dsRNA). Once inside the cell, this material is processed into short 21-26 nucleotide RNAs termed siRNAs that are used in a sequence-specific manner to recognize and destroy complementary RNA.
The report compares RNAi with other antisense approaches using oligonucleotides, aptamers, ribozymes, peptide nucleic acid and locked nucleic acid.
Various RNAi technologies are described, along with design and methods of manufacture of siRNA reagents. These include chemical synthesis by in vitro transcription and use of plasmid or viral vectors. Other approaches to RNAi include DNA-directed RNAi (ddRNAi) that is used to produce dsRNA inside the cell, which is cleaved into siRNA by the action of Dicer, a specific type of RNAse III. MicroRNAs are derived by processing of short hairpins that can inhibit the mRNAs. Expressed interfering RNA (eiRNA) is used to express dsRNA intracellularly from DNA plasmids.
Delivery of therapeutics to the target tissues is an important consideration. siRNAs can be delivered to cells in culture by electroporation or by transfection using plasmid or viral vectors. In vivo delivery of siRNAs can be carried out by injection into tissues or blood vessels or use of synthetic and viral vectors.
Because of its ability to silence any gene once the sequence is known, RNAi has been adopted as the research tool to discriminate gene function. After the genome of an organism is sequenced, RNAi can be designed to target every gene in the genome and target for specific phenotypes. Several methods of gene expression analysis are available and there is still need for sensitive methods of detection of gene expression as a baseline and measurement after gene silencing. RNAi microarray has been devised and can be tailored to meet the needs for high throughput screens for identifying appropriate RNAi probes. RNAi is an important method for analyzing gene function and identifying new drug targets that uses double-stranded RNA to knock down or silence specific genes. With the advent of vector-mediated siRNA delivery methods it is now possible to make transgenic animals that can silence gene expression stably. These technologies point to the usefulness of RNAi for drug discovery.
RNAi can be rationally designed to block the expression of any target gene, including genes for which traditional small molecule inhibitors cannot be found. Areas of therapeutic applications include virus infections, cancer, genetic disorders and neurological diseases. Side effects can result from unintended interaction between an siRNA compound and an unrelated host gene. If RNAi compounds are designed poorly, there is an increased chance for non-specific interaction with host genes that may cause adverse effects in the host.
Before performing your experiment, always make sure to work in a RNase free environment.
1. To each tube, add 250uL of the provided sterile buffer (100 mM sodium acetate, 30mM HEPES-KOH, 2mM magnesium acetate, pH 7.4) to obtain a 20 M solution
2. Heat the tube to 90°C for 2 min
3. Incubate at 37°C for 60 min
4. Perform your experiment
This procedure will disrupt higher aggregates, which may have formed in the lyphilization process.
It is necessary to ensure siRNA silencing efficiency.
Store all tubes at -20°C (dissolved or lyophilized)
You only have to perform the incubation steps (1~3) the first time you use the siRNA
The solution can be stored frozen at -20°C and freeze-thawed numerous times.