Through careful optimization--e.g. choosing the right transfection agent and transfection method--high levels of transfection efficiency can be achieved in many cell types. Once a protocol is optimized for a particular cell type, reproducible siRNA screening experiments can easily be done.

Efficient Transfection is Critical

The ability of small interfering RNAs (siRNAs) to silence gene expression is proving to be invaluable for studying gene function in cultured mammalian cells. Quite often, the success or failure of an siRNA experiment hinges on siRNA delivery. siRNAs can be transiently transfected using commonly available transfection reagents. However, high efficiency transfection of siRNA is not trivial; therefore, the utility of RNAi in difficult-to-transfect cell types may be limited.

To achieve maximum effectiveness of exogenously introduced siRNAs, transfection optimization experiments are required. Failure to optimize critical transfection parameters can render RNAi effects undetectable in cell culture. These transfection parameters include culture conditions, choice and amount of transfection agent, exposure time of transfection agent to cells, and siRNA quantity and quality. The transfection procedure itself can be a critical factor. The pre-plated transfection procedure involves pre-plating cells, meaning the cells are allowed to attach, recover, and grow for 24 hours prior to transfection. Here, we show evidence that an alternative transfection procedure, termed reverse transfection [1] or neofection [2], offers several key benefits over the traditional pre-plating method. Reverse transfection involves simultaneously transfecting and plating cells, much like procedures used for transfecting suspension cells. This method is easier and faster because it bypasses several steps of the traditional procedure. This article summarizes the use of reverse transfection to maximize performance of siRNA in cultured cells and offers suggestions on how to optimize siRNA transfection parameters.

Important Parameters in siRNA Transfection Experiments

  • Health of cultured cells
  • Transfection method
  • Transfection conditions
  • Quality and quantity of siRNA

The goal of transfection optimization is to determine the conditions that will provide maximum gene knockdown while maintaining an acceptable level of viability for the particular cell type (see sidebar, Two-Step Optimization Protocol).

Health of cultured cells.
For maximal cell viability during transfection, cells must be healthy at the beginning of the experiment--healthy cells are easier to transfect than poorly maintained cells. Overly crowded and sparse cultures are not conducive for cell health. Many cells undergo expression profile changes that can adversely affect your experiments when they are stressed by culture conditions. As a rule, cells should never be allowed to cover the entire surface area of their culture dish. Instead, cells should take up between 20 and 80 percent of the available space. Subculturing cells before they become overcrowded minimizes instability in continuous cell lines and reduces variability from experiment to experiment. Cells can gradually change in culture, and it is difficult to consistently maintain cells in perfect health; therefore, to obtain maximally reproducible experimental results, we recommend that cells be transfected within 10 passages of the optimization experiments. Cells older than this should be destroyed and replaced with new cells from a frozen stock. Finally, maintaining strict protocols, including time intervals between plating and transfecting cells, will improve experimental reproducibility.

Transfection method.
In preparation for transfection, adherent mammalian cells have been traditionally pre-plated into tissue culture wells and allowed to attach, recover, and grow for 24 h prior to transfection. Reverse transfection is an alternative method of transfection where cells are transfected while still in suspension (i.e. after trypsinization and prior to plating). The method produces equivalent or improved transfection efficiency over the standard pre-plated method for many of the cell types tested and saves an entire day in the process (Figure 1). Presumably, the amount of exposed cell surface, and not the number of transfection complexes, is the limiting factor in traditional adherent transfection. Reverse transfection is believed to increase cell exposure to transfection complexes often leading to greater transfection efficiency.


Figure 1. Standard Pre-Plated Transfection vs. Reverse Transfection with siPORT™ NeoFX™ Transfection Agent. Mammalian adherent cells are typically "pre-plated" prior to transfection, allowing them to reattach and resume growth for 24 h before exposure to transfection complexes (left). Reverse transfection or "neofection" involves adding transfection complexes to the cells while they are in suspension, prior to plating, thus saving an entire day in the transfection procedure (right).


Figure 2A shows reverse transfection of a GAPDH siRNA into seven different mammalian cell types. The data suggests that reverse transfection can deliver high levels of functional siRNA to a wide variety of cells. Some cell types transfected more efficiently by reverse transfection than the traditional method. For example, HepG2 cells, traditionally a difficult cell line to transfect, reverse transfected remarkably well (Figure 2B), perhaps because their dense growth pattern precludes adequate cell surface exposure to transfection agents once attached to a substrate.


Figure 2. Efficient Reverse Transfection of Various Cell Lines. (A) Reverse transfection of a GAPDH siRNA (Silencer® GAPDH siRNA, Ambion) into seven different mammalian cell types. Amounts of siPORT™ NeoFX™ (Ambion), siRNA amounts, and cell density were optimized for each cell line (data not shown). All cells were harvested and analyzed by real-time RT-PCR for GAPDH mRNA levels at 48 hours after transfection. (B) HepG2 and HeLa cells were both reverse transfected during plating and transfected after pre-plating the cells with an siRNA targeting GAPDH (Silencer® GAPDH siRNA, Ambion) or Negative Control siRNA (Silencer® Negative Control #1 siRNA, Ambion). 48 hours post-transfection, GAPDH expression was measured by real-time RT-PCR. Percent gene expression was calculated as GAPDH gene expression in GAPDH siRNA transfected cells compared to those transfected with the Negative Control siRNA.


Cell density became a less critical parameter, requiring little to no optimization, when cells were reverse transfected. Figure 3A demonstrates that a broad range of cell concentrations were reverse transfected efficiently, whereas traditional pre-plated transfections required careful optimization of cell density (Figure 3B). In addition, reverse transfection is faster--a full day can be saved because cells do not have to be plated prior to transfection. Because of these fundamental advantages, Ambion scientists routinely optimize transfection of new cell lines using the reverse transfection procedure.


Figure 3. Reverse Transfection Yields Higher Tolerance to Cell Plating Density. (A) SKOv3 cells were reverse transfected in a 96 well plate using 10 nM and 30 nM GAPDH siRNA (Silencer® GAPDH siRNA, Ambion) or Negative Control siRNA (Silencer® Negative Control #1 siRNA, Ambion) at the indicated cell densities using siPORT™ NeoFX™ Transfection Agent (0.3 µl per well, Ambion). At 48 hours post-transfection, cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. Remaining gene expression was calculated as a percentage of GAPDH mRNA in cells transfected with GAPDH siRNA compared to cells transfected with Negative Control siRNA. Data were normalized against the 18S rRNA signal. (B) COS-7 cells were pre-plated in a 24 well dish at the indicated plating densities 24 hours prior to transfection. Transfections were performed with either 10 nM GAPDH or Negative Control #1 siRNA using siPORT™ Amine Transfection Agent (4 µl per well, Ambion). Remaining gene expression was determined as described for Panel A.


Transfection conditions.
Overall, transfection efficiency and cell viability are dependent on choice and amount of transfection agent and exposure time of cells to transfection agent. Commercially available reagents perform with varying levels of effectiveness depending on the cell type. A successful match between cell line and reagent can usually be made by testing several commercially available agents. Transfection agent volume is also critical--too little will not transfect efficiently; too much can be cytotoxic. Both siRNA transfection efficiency and cell viability should be considered when designing transfection agent screening experiments. The ideal reagent is one that yields effective target gene reduction without significant cell mortality. In HepG2 cells, siPORT™ NeoFX™ Transfection Agent shows minimal toxicity and yields a broad range of silencing activity (Figure 4). Some reagents and cell lines are not as flexible and require more precision. Ambion recommends testing transfection agents that have been validated specifically for siRNA transfection. We have found that most DNA-based transfection agents are ineffective for siRNA delivery.


Figure 4. Transfection Agent Cytotoxicity. Multiple siPORT™ NeoFX™ Transfection Agent volumes (0.03-1.0 µl, Ambion) were used in reverse transfection of HepG2 cells. Assays were done in 96 well plates with 5 nM GAPDH siRNA (Silencer® GAPDH siRNA, Ambion) or Negative Control siRNA (Silencer® Negative Control #1 siRNA, Ambion). Remaining gene expression (bars) was determined as described for Figure 3A. Cell viability (line) was also measured using the ViaCount® Assay (Guava Technologies). As reagent volume increased, greater levels of siRNA-mediated reduction of target gene expression were obtained. In this cell type, siPORT NeoFX shows minimal toxicity and yields a broad range of silencing activity. Some reagents are not as flexible and require more precision.


Length of cell exposure to transfection agents should be optimized to minimize cellular toxicity and maximize siRNA activity by varying the amount of transfection agent and cell exposure time to transfection complexes (Figure 5). Media containing transfection agent was removed from the wells at the indicated time points and replaced with fresh media. Cellular viability, apoptosis, and siRNA activity were measured 48 hours after addition of 1 or 2 µl transfection agent + GAPDH siRNA or negative control siRNA. In wells where transfection agent was not removed, cells appeared necrotic, they underwent moderate levels of apoptosis, and cell viability was >30% less than a nontreated control well. These same samples, however, showed >90% reduction in GAPDH gene expression over negative control wells. When transfection complexes were removed at 4 hours post transfection, cell viability was >95%, apoptosis was minimal, but GAPDH silencing was 30% less than in cultures experiencing no media change. When cells were exposed to 1 µl transfection agent for 24 hours post-transfection before a media change, GAPDH silencing was high, comparable to cells with no media change, and cell viability was nearly 90%. These data suggest that careful optimization of cell exposure to transfection complexes can improve the quality of data generated in RNAi experiments.


Figure 5. Exposure Time to Transfection Complexes. HeLa cells (5 x 103 cells/well) were exposed to transfection complexes containing one of two concentrations of transfection agent (1-2 µl) + 10 nM GAPDH (Silencer® GAPDH siRNA, Ambion) or negative control siRNA (Silencer® Negative Control #1 siRNA, Ambion). Medium was changed to remove transfection complexes at different time points. Cellular viability, apoptosis, and siRNA activity were measured 48 h after transfection began. Cell viability (blue line) was measured using the ViaCount® Assay (Guava Technologies). Apoptosis (yellow line) was measured using a Guava® PCA™-96 instrument (Guava Technologies). Remaining gene expression (green bars) was determined as described for Figure 3A. NT = Not Transfected


 Quality and quantity of siRNA.
The quality and quantity of siRNA used for transfection significantly influences RNAi experiments. siRNA should be free of contaminants carried over from synthesis including salts, proteins, and ethanol. Additionally, the siRNA should also be <30 bp, because the presence of dsRNA larger than approximately 30 bp has been shown to alter gene expression by activating the nonspecific interferon response [3].

The optimal concentration of siRNA is influenced by several factors including properties of the target gene and cell type. As mentioned above, too much siRNA may lead to off-target effects; too little can result in undetectable gene silencing. In general, 1-30 nM siRNA is a good concentration range within which to optimize transfection (10 nM is a sufficient starting point). In Figure 6, transfection of HeLa cells was optimized at very low concentrations of siRNA. HeLa cells are easier to transfect than many other cell types, and 10 nM siRNA in combination with reverse transfection is sufficient for obtaining optimal target gene reduction.



Figure 6. Optimal Amount of siRNA. HeLa cells were split and resuspended in growth media at 4.0x104 cells/ml. Transfection complexes were prepared containing the indicated concentration of chemically synthesized GAPDH siRNA (Ambion) or Negative Control siRNA #1 (Ambion; data not shown), and 0.3 µl siPORT™ NeoFX™ Transfection Agent (Ambion). 48 hours post-transfection, cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. Remaining gene expression was determined as described for Figure 3A.

Conclusion

There is no single transfection parameter that by itself ensures efficient siRNA uptake by cells in culture. Optimal siRNA uptake into viable cells is achieved by systematically addressing each of several critical variables. Ambion provides a series of tools to simplify RNAi experiments including two transfection agents designed specifically for siRNA delivery. Both siPORT NeoFX and siPORT Amine Transfection Agents can be used for reverse transfection or standard transfection of siRNAs into a wide variety of cell types. The siPORT Transfection II Kit contains samples of both of these reagents as well as positive and negative control siRNAs for protocol optimization. Ambion's siRNA Delivery Resource has further details on transfection optimization, recommendations for transfection agent, and conditions to use with specific cell types.

siPORT™ Amine is manufactured for Ambion by Mirus.

Scientific Contributors
Rich Jarvis • Ambion, Inc.