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.
RNAi workflow
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
Handling RNA
RNA oligonucleotides are susceptible to degradation by exogenous ribonucleases introduced during handling.
- Wear gloves when handling RNA.
- Use RNase-free reagents, tubes, and barrier pipette tips for preparing RNA for transfection.
- Work areas should be wiped down with 70% ethanol or other RNase decontamination solution such as RNaseZap RNase Decontamination Solution.
Transfection efficiency
The efficiency with which mammalian cells are transfected with siRNA will vary according to cell type and the transfection agent used. This means that the optimal concentration used for transfections should be determined empirically. The major variables that impact siRNA transfection efficiency are the following:
- Transfection reagent type and amount
- Number of cells plated in well
- Type of RNA or siRNA
- Concentration of RNA or siRNA
Positive controls
It is important to include a positive control in each experiment. The positive control should elicit a reproducible, easily measured response in the cells and assay used in your study. If you see maximal effect above/below a pre-determined threshold level with this control, you know that measurements from other experiments tested on the same day are reliable. Note that it is important to empirically determine the thresholds for each assay and control pair.
The degree of the response to a particular RNA or siRNA is directly linked to its transfection efficiency. To assess transfection efficiency, we recommend including the BLOCK-iT Fluorescent Oligo in every experiment. Using the BLOCK-iT Fluorescent Oligo in your transfection experiment allows you to easily assess oligomer uptake and transfection efficiency using any fluorescence microscope and a standard FITC filter set. Uptake of the fluorescent oligomer by at least 80% of cells correlates with high efficiency.
Negative controls
Negative controls are just as important as positive controls for obtaining meaningful data. Always include a set of transfections with an equimolar amount of at least one negative control to compare the effects of the target RNA or siRNA-treated and control treated cells. Data from these crucial controls serve as a baseline for evaluation of experimental target knockdown.
Non-transfected or cells-only negative controls are also very useful. By comparing expression of a housekeeping gene among cultures that were not transfected and cultures transfected with a non-targeting negative control, valuable information about the effects of transfection on cell viability can be obtained.
| Type of control | Recommended use | Recommended products |
|---|---|---|
| Transfection control | Calculate and monitor transfection efficiency with fluorescence |
|
| Negative control | Nonspecific or scrambled controls used to measure knockdown levels vs. background |
|
| Positive control | RNAi reagents known to achieve high levels of knockdown used to measure delivery and optimize experimental conditions |
|
| Untransfected control | Measure normal gene expression level and phenotype | |
| Multiple RNAi sequences to the same RNAi target | Use to verify phenotypic change, control for off-target effects for generating publication quality results | |
| Titration of RNAi | Use the lowest effective level to avoid altering the cells normal processes | |
| Rescue experiments | Turn off inducible RNAi or introduce a plasmid expressing the target mRNA that the RNAi sequence will not affect | BLOCK-iT Pol II miR RNAi or BLOCK-iT shRNA vectors with inducible promoters (CMV/TO and H1/TO respectively) |
Co-transfection
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.
siRNA quality
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.
Store siRNAs at –20°C or –80°C, but do not use a frost-free freezer. Our data indicate that up to 50 freeze/thaw cycles are not detrimental to siRNAs in solution at 100 μM (as assessed by mass spectrometry and analytical HPLC). However, we recommend that siRNAs that have been resuspended in RNase-free water or buffer be stored in small aliquots to avoid potential contamination.
Annealed, double-stranded siRNAs are much more nuclease resistant than single-stranded RNA. However, stringent RNase-free techniques should be used during all RNAi experiments.
If you suspect that a preparation of siRNA may be degraded, check the integrity of the siRNA by running ~2.5 μg on a non-denaturing 15–20% acrylamide gel. Visualize the RNA by staining with ethidium bromide, and verify that it is the expected size and intensity. The siRNA should migrate as a tight band; smearing indicates degradation.
siRNA quantity
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.
Volume of transfection reagent
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.
Cell density
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.
Exposure to transfection agent/siRNA complexes
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.
Presence of serum during transfection
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.
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