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?

In readily transfected cells treated with potent and effective siRNAs such as the new Ambion Silencer Select siRNAs, near-maximal silencing can be achieved for 5–7 days. Figure 1 shows data from a representative experiment. Four different Silencer Select Pre-designed siRNAs were transfected at 5 nM into HeLa cells. Maximal knockdown (>85%) was seen on day 2 post-transfection and knockdown persisted at >80% through days 5–7. Significant knockdown was still observed at day 10 with all but one siRNA. It should be noted that HeLa cells fall into an easy-to-transfect category, and good knockdown (without compromising cell viability) was achieved at low transfection agent concentrations of 0.15 µL/well (96-well format). A similar duration of siRNA-induced silencing was observed in BJ cells (Figure 2), but somewhat higher concentrations of transfection agent were required to achieve efficient delivery (0.3 µL/well).


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?

No, raising the siRNA concentration from 5 nM to 50 nM did not improve or prolong silencing (Figure 2). Presumably, the RISC is saturated upon efficient transfection of a highly potent siRNA at 5 nM; excess siRNA could potentially be rapidly degraded, sequestered, or excreted from the cell. This result is consistent with a previous study that reported effective silencing (>80% knockdown) by Silencer Select siRNAs at both high (100 nM) and low (3 nM) concentrations [1]. The authors further showed that reduced siRNA concentrations resulted in fewer off-target effects on gene expression, leading to improved knockdown specificity.


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?

Repeated transfections can prolong silencing in some cases, however, we have seen variable results with this approach. Figure 3 shows that with 3 of 4 siRNAs tested in HeLa cells, adding a second siRNA transfection at day 4 resulted in improved knockdown at days 6–11 compared to the single transfection, although only one of the 4 siRNAs achieved >70% knockdown out to day 14. When the second transfection was performed 24 hours after the first transfection, no significant improvement of the longevity of the RNAi effect was observed (data not shown). The results show that repeated siRNA transfection leads to improved knockdown for some siRNAs at day 6 and beyond, but that knockdown levels may only rarely reach the maximal levels seen at day 2.


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.

Conclusions

In our experiments, a single transfection of 5 nM Silencer Select siRNAs achieved >80% knockdown that lasted 5–7 days post-transfection, then progressively diminished. Higher siRNA concentrations did not result in stronger or longer-lasting knockdown, but are likely to cause off-target effects. In some cases, repeated siRNA transfection resulted in improved knockdown during select timepoints post-transfection.

Scientific Contributors:
Mu Li, Angie Cheng, Alexander (Sasha) Vlassov, Susan Magdaleno • Applied Biosystems Inc., Austin, TX