RNA interference (RNAi) is an evolutionarily conserved mechanism for silencing gene expression
Chemically synthesized short interfering RNAs (siRNAs) can be used to induce silencing in cultured mammalian cells. Upon cellular uptake, the guide strand of the siRNA is incorporated into an RNA-induced silencing complex (RISC) that directs degradation of messenger RNAs containing complementary sequences. siRNAs are extensively used as a tool to understand gene function in cellular pathways in healthy and diseased cells and animals, and they have tremendous potential for therapeutic gene silencing. Here we answer questions regarding the persistence of the RNAi effect initiated by transfecting siRNAs into human cells.
Recently, Applied Biosystems launched a next generation siRNA product, Ambion® Silencer® Select siRNAs. These siRNAs are designed using a new algorithm that improves silencing efficiency and potency, and they are chemically modified with locked nucleic acids (LNAs) to improve specificity and reduce off-target effects. Applied Biosystems scientists investigated the duration of silencing induced by these siRNAs in two cell lines, and studied the effects of siRNA concentration and repeated transfections on silencing duration.
What is the duration of silencing after a single siRNA transfection?
Figure 1. Duration of Silencing After a Single Silencer® Select siRNA Transfection. HeLa cells (500 cells/well) were transfected with four different Ambion® Silencer Select Pre-designed siRNAs at 5 nM. Cells were lysed day 1 through 10 post-transfection as indicated using the TaqMan® Gene Expression Cells-to-CT™ Kit, and real-time RT-PCR was performed directly in the cell lysates using the indicated TaqMan Gene Expression Assays. Knockdown data is expressed relative to data from cells transfected with Ambion Silencer Select Negative Control #1 siRNA.
Can silencing be prolonged by raising the siRNA concentration transfected?
Figure 2. Longevity of the RNAi Effect at Two Different siRNA Concentrations. BJ cells (500 cells/well) were transfected with the same Ambion® Silencer® Select Pre-designed siRNAs as described in Figure 1 at either 5 nM or 50 nM siRNA. Knockdown was measured as outlined in Figure 1.
Can prolonged silencing be achieved by repeated siRNA transfections?
Figure 3. Duration of Silencing After Single vs. Double Transfections. BJ cells (500 cells/well) were transfected with four independent Ambion® Silencer® Select Pre-designed siRNAs (5 nM). Single transfection at day 0; Double transfection at days 0 and 4 (indicated with arrow). Knockdown was measured as outlined in Figure 1.
Mu Li, Angie Cheng, Alexander (Sasha) Vlassov, Susan Magdaleno • Applied Biosystems Inc., Austin, TX