Small interfering RNA (siRNA) transfectionTransfection protocol Download
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Small interfering RNA (siRNA) contains 21-25 nucleotides, specific for target RNA. Once entry into cells, siRNAs can be recruited into a multi-protein complex, known as RNA induced silencing complex (RISC), which interacts with the target RNA to mediate mRNA degradation, thus knock-down or suppress the expression of target gene . siRNA transfection can be carried out with non-viral methods or viral vectors containing short hairpin RNA (shRNA), which can be subsequently processed into active siRNA.
Figure 4. The biogenesis and function of miRNAs .
siRNA transfection in vitro
siRNA transient transfection in vitro can be mediated by polymer and liposome. Stable transfection cell lines need to be mediated by viral vectors (lentivirus, adenovirus, AAV) carry shRNA targeting gene of interest. The knock-down of shRNA varies in different sequence and needs to be tested. Besides, shRNA should be cloned into viral vectors for virus production. Therefore, viral-based stable transfection takes so much time but presents little cytotoxicity and high efficiency.
siRNA transfection in vivo
Due to the instability of siRNA, low delivery efficiency and the superior difficulties in specific tissue distribution, in vivo siRNA transfection presents great technical challenges. Three methods have been adapted to deliver siRNA in vivo: 1) siRNAs with chemical modifications are generated, such as morpholino (MO) , 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA) , 2'-deoxy-2'-fluoro and 2'-O-methyl ribosugar modifications , Locked Nucleic Acid (LNA) modifications ; 2) encapsulation into liposomes also presents great protection from degradation in serum ; 3) viral vectors (lentivirus, adenovirus, AAV) containing shRNA targeting gene of interest show much higher delivery efficiency in vivo than chemical modifications or encapsulation, but these methods are time-consuming and might show some immunogenicity.
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