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.

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).

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
(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.