siRNA transfection is a powerful tool to understand underlying mechanisms in gene regulation and molecular pathways. Transfecting siRNA with a high efficiency while minimizing off-targets and side effects will not be a challenge for you anymore thanks to these tips.
- Use the most appropriate siRNA concentration. Using low siRNA concentrations such as 1–5 nM avoids off-target effects and unwanted toxic side effects. Use a transfection reagent suitable for low-siRNA concentrations. The siRNA final concentration may nevertheless range from 1 to 50 nM depending on the cells and the target gene.
- Prepare a suitable siRNA stock solution. When using low siRNA concentrations, adapt the concentration of the siRNA stock solution to pipet accurate volumes. Proceed the same way for small volumes of transfection reagent: dilute 1 to 5 in sterile water.
- Transfect healthy cells. Passage cells at least twice after thawing to allow recovery before transfection, and use cells at low passage number (< 20 passages). Discard overconfluent cells. Regularly check for mycoplasma contaminations. Seed cells the day before transfection according to the confluency recommended by the transfection reagent provider.
- Check serum quality. Some serum lots may drastically inhibit transfection efficiency, hence resulting in lower silencing efficiency. Check transfection efficiency of different serum lots before purchasing a new batch of serum.
- Know the target gene in and out. Design the siRNA sequence as efficiently as possible by using several algorithms. The better the siRNA, the lower the concentration needed for high silencing. Check the half-lives of the protein and mRNA of interest and measure gene silencing accordingly 24 to 96 hours after transfection. Analyze gene silencing at both mRNA and protein levels.
- Always use positive and negative controls. Use an siRNA against a housekeeping gene (GAPDH, cyclophylin B) as a positive control. Use a commercially available negative control (mismatch, non-targeting). Avoid fluorescent siRNA controls when working at low-siRNA concentration since high-siRNA concentration is required for detection (20–50 nM).
- Follow up the transfection reagent protocol. Some reagents are inhibited by serum or antibiotics, while others may be used in serum- and antibiotic-containing medium, hence reducing the risks of toxicity and the number of steps in the protocol. Check the recommended cell confluency.
- Perform an appropriate read-out. Test different antibodies if possible. Ensure that all isoforms of the targeted protein are recognized by the antibody used. Measure gene silencing at different times after transfection to find the optimal window.
- Store the reagents appropriately. Store the transfection reagent at 4°C upon reception; aliquot the siRNA and store at -20°C.
Want more guidance? Check out this video, which walks you through the siRNA transfection workflow.