Guidelines for RNA Transfection

Transfection of RNA is an offshoot of classic transfection technologies for introducing RNA into cells. The purpose of RNA transfection is similar to that of plasmid transfection. mRNA is introduced into cells to express the encoded protein, and study gene function and regulation. siRNA is used for RNAi studies that examine the effects of gene knockdown. One major difference between the two methods is that RNA can only be transiently transfected.

The diagram below depicts an RNAi experiment workflow following siRNA design and synthesis. When performing an RNAi experiment, make sure that you have the following on hand:

• Transfection/electroporation agent and protocol
• Assays to assess knockdown and other RNAi effect(s)
• Positive and negative control siRNAs
• Two or more siRNAs to gene of interest

Figure 1: RNAI workflow following siRNA design and synthesis

Co-transfection is performed when the user wants to introduce both siRNA and a plasmid for expressing a protein into a cell. This protein can be part of the test system, or in most cases, it can be a reporter gene (luciferase, GFP, β-lactamase). In some cases, users may want to express a mutant protein along with the siRNA to block one pathway with the siRNA, and overexpress a mutant protein.

The presence of the plasmid may decrease transfection efficiency of all cargo (plasmid and siRNA) when a lipid transfection reagent is used, making transfection optimizations very important. Undesired and non-specific cell death can result with too much lipid, or too little knock-down or protein expression from the plasmid can occur if transfection conditions are not optimal.

The quality of siRNA can significantly influence RNAi experiments. siRNAs must be free of reagents carried over from synthesis, such as ethanol, salts, and proteins. Also, dsRNA contaminants longer than 30 bp are known to alter gene expression by activating the nonspecific interferon response and causing cytotoxicity (Stark et al., 1998). Therefore, we recommend using standard purity siRNAs that are greater than 80% full length.

The optimal amount of siRNA and its capacity for gene silencing are influenced in part by properties of the target gene products, including the following: mRNA localization, stability, abundance, as well as target protein stability and abundance.

Although many siRNA experiments are still performed by transfecting cells with 100 nM siRNA, published results indicate that transfecting lower siRNA concentrations can reduce off-target effects exhibited by siRNAs (Jackson et al., 2003; Semizarov et al., 2003). For lipid-mediated reverse transfections, 10 nM of siRNA (range 1–30 nM) is
usually sufficient. For siRNA delivery using electroporation, siRNA quantity has a less pronounced effect, but typically 1 μg/50 µL cells (1.5 μM) of siRNA (range 0.5–2.5 μg/50 µL cells or 0.75–3.75 μM) is sufficient.

Keep in mind that while too much siRNA may lead to off-target or cytotoxic effects, too little siRNA may not reduce target gene expression effectively. Because there are so many variables involved, it is important to optimize the siRNA amount for every cell line used. In addition, the amount of non-targeting negative control siRNA should be the same as the experimental siRNAs.

The volume of transfection agent is a critical parameter to optimize because too little can limit transfection, and too much can be toxic. The overall transfection efficiency is influenced by the amount of transfection agent complexed to the siRNA. To optimize, titrate the transfection agent over a broad dilution range, and choose the most dilute concentration that still gives good gene knockdown. This critical volume should be determined empirically for each cell line.

While cell density is important for traditional, pre-plated transfection experiments, cell density is less critical and requires little to no optimization, when siRNAs are delivered by reverse transfection. However, if too many cells are used, and the amount of siRNA is not increased proportionally, the concentration of siRNA in the sample may be too low to effectively elicit gene silencing. When cell density is too low, cultures can become unstable. Instability can vary from well to well because culture conditions (e.g., pH, temperature) may not be uniform across a multiwell plate and can differentially influence unstable cultures.

Although most transfection agents are designed to induce minimal cytotoxicity, exposing cells to excessive amounts of transfection agent or for an extended time can be detrimental to the overall health of the cell culture. Sensitive cells may begin to die from exposure to the transfection agent after a few hours. If transfection causes excessive cell death with your cells, remove the transfection mixture and replenish with fresh growth medium after 8–24 hours.

Complex formation between transfection agents and siRNA should be performed in reduced-serum or serum-free medium, so that serum components will not interfere with the reaction. However, once complex formation has occurred, some transfection agents will permit transfection in serum-containing, normal growth medium (follow manufacturer’s instructions). No culture medium addition or replacement is usually required following transfection, but changing the media can be beneficial in some cases, even when serum compatible reagents are used. Be sure to check for serum compatibility before using a particular agent. Some transfection agents require serum free medium during the transfection and a change to complete growth media after an initial incubation with transfection complexes.