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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.
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 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.
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.
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.
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.
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.
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.
For Research Use Only. Not for use in diagnostic procedures.