Intracellular siRNA Delivery For In Vitro Gene Silencing

One of the major challenges in the field of RNA interference is the intracellular delivery of siRNA. Normally, the cell membrane is impermeable to nucleic acids. However, various techniques have been developed to transport the siRNA into the cell. Most of these techniques have one of the following drawbacks: toxicity to the cells, low efficiency, little release into the cytoplasm, or degradation of the siRNA.

The SAINT-RED reagent developed by Synvolux Therapeutics has neither of the afore mentioned draw backs. This reagent is based on a cationic pyridinium amphiphile combined with a helper lipid. The formulation has been optimized for the intracellular delivery of siRNA into the cytoplasm of eukaryotic cells. Furthermore, the performance has been compared to various competitors and found to be superior. And last but not least the SAINT-RED reagent has proven to protect the siRNA from degradation.

Figure 1 Remaining CD326 expression 72 hours after transfection with siRNA.

A siRNA sequence against the CD326 cell surface protein was transfected using 5 different transfection reagents: SAINT-RED and 4 competitors A to D. SKOV-3 cells were plated in 96-well plates and transfected the next day according to the manufacturers protocol. Knockdown efficiency was measured after 96 hours by incubation with an antibody-Alexa488 conjugate against CD326. Percentages CD326 expression were normalized to the no-siRNA control (set 100%, not shown). SAINT-RED exhibits the most effective inhibition of CD326 expression (85%-90%). Competitor D reaches almost the same level of knock down of gene expression, however 5 times as much siRNA is required compared to SAINT-RED. This comparison illustrates that SAINT-RED is one of the most efficient reagents on the market for siRNA transfections.

In another experiment the stability of siRNA complexed with SAINT-RED was studied. siRNA against a housekeeping gene was complexed with SAINT-RED and stored at 4°C. Simultaneously a solution with only siRNA was stored at 4°C as a control. After various time intervals samples were taken of these stored solutions, and transfections were performed. The inhibition of the housekeeping gene expression was measured 48 hours after transfection. The results of this experiment are shown in figure 2.

Figure 2 siRNA stabilization by complexation with Saint-RED.

From these results it is clear that SAINT-RED stabilizes siRNA when complexed together.

Finally, some proteins show a delayed response when knocked down with siRNA as only the mRNA is targeted and not the proteins directly. This delayed response is caused by the life time of the particular protein. The example shown in figure 3 shows once more the potency of the SAINT-RED reagent for delivering siRNA to living cells to knock down a gene of choice. In this experiment two siRNA's against different target sequences of the same gene were tested: siRNA2 and siRNA3. The cells were treated only once with SAINT-RED/siRNA complex on day zero and the gene expression was monitored for two weeks. The resulting down regulation shows remarkable similarity for both siRNA's and in this particular case the maximum effect is reached after 12 days.

Figure 3 Prolonged down-regulation after transfection with Saint-RED/siRNA

To summarize the results, SAINT-RED is a powerful siRNA transfection reagent. The time frame in which maximum down regulation is accomplished varies for different target proteins depending on the half-life of the particular protein. The fact that the siRNA is protected from degradation when complexed with SAINT-RED is one of the unique features of this easy-to-use delivery reagent. Therefore, it is highly recommended as a research tool for anyone in the field of molecular cell biology who needs to knock down a particular gene.