Guidelines for transfection
Guidelines for transfection of siRNA
The application of RNA interference (RNAi) to mammalian cells has revolutionized the field of functional genomics. The ability to simply, effectively, and specifically downregulate the expression of genes in mammalian cells holds enormous scientific, commercial, and therapeutic potential. Efficient transfection of siRNA is critical for effective gene silencing.Off-target effects in RNAi experiments
Studies have indicated that transfection of siRNA can result in off-target effects, in which siRNAs affect the expression of nonhomologous or partially homologous gene targets. Off-target effects can include mRNA degradation, inhibition of translation, or induction of an interferon response (5–8). The mechanisms of off-target effects are not fully understood. They may be caused by siRNA targeting mRNA with close homology to the target mRNA, by siRNAs functioning like miRNAs, or by a cellular response to siRNA toxicity. In addition, some researchers have observed an siRNA-mediated interferon response.
Research suggests that off-target effects, which may produce misleading results in RNAi experiments, can be largely avoided by using low siRNA concentrations (9, 10).
Optimizing siRNA transfection
Calculating concentrations of siRNA
Approximate values for a double-stranded, 21 nt siRNA molecule:
- 20 µM siRNA is equivalent to approximately 0.25 µg/µl
- The molecular weight of a 21 nt siRNA is approximately 13–15 µg/nmol
To achieve the best results in siRNA transfection of adherent cells, we recommend optimizing the following parameters.
Amount of siRNA
The amount of siRNA used is critical for efficient transfection and gene silencing. The ratio of transfection reagent to siRNA should be optimized for every new cell type and siRNA combination used.
Cell density at transfection
The optimal cell confluency for transfection should be determined for every new cell type to be transfected and kept constant in future experiments. This is achieved by counting cells before seeding and, in the case of using a traditional protocol, by keeping the interval between seeding and transfection constant. This ensures that the cell density is not too high and that the cells are in optimal physiological condition at transfection.
A guide to the number of cells to seed for different formats is shown in the table Typical number of adherent cells to seed.
Typical number of adherent cells to seed
| Culture format | Fast-forward or reverse-transfection (day of transfection) |
Traditional protocol (day before transfection) |
|---|---|---|
| 384-well plate | 4000–10,000 | 2000–5000 |
| 96-well plate | 1–5 x 104 | 0.5–3 x 104 |
| 48-well plate | 2–8 x 104 | 1–4 x 104 |
| 24-well plate | 0.4–1.6 x 105 | 2–8 x 104 |
| 12-well plate | 0.8–3 x 105 | 0.4–1.6 x 105 |
| 6-well plate | 1.5–6 x 105 | 0.8–3 x 105 |
| 60 mm dish | 0.3–1.2 x 106 | 1.5–6 x 105 |
| 100 mm dish | 2–4 x 106 | 1–2 x 106 |
Transfection in multiwell plates — preparing a master mix
If you are performing transfection in multiwell plates, prepare a master mix of transfection complexes or of transfection reagent and culture medium (depending on the protocol) for distribution into plate wells.
- Calculate the required volumes of each component and the total volume before you prepare the master mix.
- Prepare 10% more master mix than is required to allow for pipetting errors (i.e., for a 48-well plate, prepare enough master mix for 53 wells).
- Use a repeat pipet to distribute the master mix.