Rich Jarvis (Associate Scientist, Ambion, Inc.)
Alan D. King (Vice President and CSO, Cyto Pulse Sciences, Inc.)
Low transfection efficiency is the most frequent cause of unsuccessful gene silencing experiments. We have found that by optimizing a few critical variables, higher levels of transfection efficiency can be achieved in many cell types.
Good 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. siRNAs or DNA constructs that express siRNAs can be transiently transfected into mammalian cells using standard techniques. However, obtaining efficient siRNA delivery by chemical transfection or electroporation is not trivial and may limit the utility of RNAi in some cell types.
A variety of methods have been developed for transfecting nucleic acids into cultured cells. The choice between chemical transfection or electroporation is largely dependent on the cell type and the characteristics of the nucleic acid being transfected. Some cell types, including most immortalized cell lines, can be transfected with any number of commercially available chemical transfection agents with careful optimization. Other cell types, such as some finite cell lines or freshly isolated primary cells, present more of a challenge. Recent publications have described the use of electroporation (1) and viral-mediated methods (2) for high efficiency transfer of nucleic acids. For some primary cell types, these methods are well-developed and offer high efficiency delivery of nucleic acid to cells in culture. However, there is still much to be learned before transfection of siRNAs into primary cells becomes a simple exercise.
Optimizing chemical transfection. Chemical transfection reagents such as cationic liposomal and polyamine based agents are commonly used to facilitate transfection of RNA, siRNA, DNA plasmids, or PCR products into cultured mammalian cells (3-6) This is, in part, due the relatively low cost and wide availability of these agents. In order to achieve maximum effectiveness of exogenously introduced nucleic acid with these agents, transfection optimization experiments are required. These experiments can be time-consuming and difficult without the correct approach. Electroporation of siRNA typically requires optimization of various parameters that affect cell uptake and cell viability.
This article will attempt to shed light on some common pitfalls and provide suggestions for maximizing the transfection efficiency of siRNA, siRNA expression plasmids, and siRNA expression PCR products in tissue culture. Some critical transfection parameters, such as cell culture conditions, the choice, amount, and use of transfection agents, and quantity and quality of nucleic acid will be discussed.
Important Parameters in siRNA Chemical Transfection Experiments
Whether you are transfecting RNA, siRNA, DNA plasmids, or PCR products, similar rules apply. When seeking optimal transfection conditions there are a few things to keep in mind. The two most important parameters that will determine the outcome of any transfection are cell health and confluency, and agent compatibility.
Cell line maintenance. Healthy cells transfect better than poorly maintained cells. Routinely subculturing cells before they become overcrowded or unhealthy will minimize experimental variability in continuous cell lines. Since cells may gradually change in culture, using cells within a defined passage number and adhering to strict protocols, including parameters for intervals between plating and transfecting cells, will improve experimental reproducibility.
Another important factor for obtaining high transfection
efficiency is cell density. For most adherent cells, the
optimal confluency for transfection is between 30-70% at the
time of transfection. Suboptimal cell
density whether too low or too high can result in poor uptake of the nucleic acid:transfection agent complexes and thus, insufficient silencing of the gene of interest. Since transfection efficiency of suspension cells is less dependent on cell density than adherent cells, instead of using the standard technique of cell plating hours before the transfection, cells can be simultaneously plated and transfected. For many cell lines, plating the cells and adding transfection complexes at the same time can be beneficial. It saves time and in some instances improves transfection efficiency. At Ambion, we routinely transfect both cell lines and primary cells using this method.
Choice of transfection agent. Not all agents are created equal. Some DNA transfection agents are incompatible with RNA. Further, agents meant for the transfection of large nucleic acids may not efficiently transfect smaller molecules. It is therefore best to try different transfection agents and cell culture conditions to determine what works best for your system.
* For special cell types that are difficult to transfect, different types of transfection agents (e.g. liposomal vs. polyamine formulations) should be tested. The volume is critical -- transfection is inefficient with too little agent, and too much can be cytotoxic.
Quality and Quantity of siRNA. The quality and quantity of the nucleic acid significantly influences siRNA transfection. Nucleic acid should be free of contaminants carried over from its preparation. Small siRNA synthesis reactions may be contaminated with salts, proteins, or ethanol. In addition, the presence of dsRNA larger than approximately 30 bp has been shown to activate the nonspecific interferon response in mammalian cells (7). Plasmid DNA preparations must be free from contaminating nucleic acid and bacterial components such as endotoxins. The optimal concentration of the nucleic acid is influenced by several factors including properties of the target gene, cell type, and the nature of the agent used. Too much nucleic acid may result in cytotoxicity or poor complexing with the transfection agent. Conversely, if too little is transfected, gene target knock down may be undetectable. Concentrations of siRNA typically used for transfection range from 1 nM to 100 nM or more. However, it should be noted that the higher the concentration, the more likely one will see off-target effects (see Designing Controls for siRNA Experiments for a more detailed discussion). Transfections of plasmids or PCR fragments usually use from 50 ng to several microgams of nucleic acid, depending on the size of the reaction.
siRNA Experiments Addressing Transfection Variables
Several experiments were designed to test transfection conditions using siRNAs as the nucleic acid transfectant. Transfections were performed using Ambion's siPORT Amine Transfection Agent. Cells were typically plated 24 hours prior to transfection so that they were healthy and growing. Some reagents require that transfections be performed in serum-free media, and that the transfection complexes be added or removed after several hours. With siPORT Amine, however, cells can be easily transfected with or without pre-plating, and in the presence or absence of serum. For each experiment, siPORT Amine was added to pre-plated COS-7 cells one day before transfection. Approximately 48 hrs after transfection, total RNA from the cell cultures was extracted using Ambion's RNAqueous-4PCR Kit, and quantitated. The expression level of both target and control genes was determined for each sample using either Northern analysis (NorthernMax-Gly Kit, Ambion) or real-time RT-PCR with gene specific primers and TaqMan probes (Applied Biosystems). For real-time RT-PCR assays, transfections were performed in duplicate or triplicate and all samples were normalized to 18S rRNA levels.
Effect of cell density on transfection of siRNA. To test whether cell density at the time of transfection affects siRNA-mediated mRNA reduction, COS-7 cells were plated in a 24 well dish at different plating densities 24 hours prior to transfection (Figure 1). Transfections were performed with either 10 nM GAPDH siRNA or with a negative control (scrambled) siRNA using 4 l of siPORT Amine Agent. After 48 hr mRNA expression was evaluated by real-time RT-PCR. Reduction in gene expression varied significantly at different cell plating densities. GAPDH siRNA-mediated reduction in mRNA was maximal at relatively low plating densities between 2.5 and 5 x 104 cells per well; little to no mRNA reduction was observed at higher plating densities .
Effect of transfection agent on siRNA activity. To study the effects of increasing amounts of transfection agent on siRNA activity, COS-7 cells (3 x 104 cells/well) were plated into a 24 well plate 24 hr prior to transfection. Cells were transfected using different volumes of siPORT Amine Transfection Agent as shown in Figure 2. mRNA expression was evaluated by real-time RT-PCR approximately 48 hrs after transfection. The data indicate that increasing amounts of transfection agent (from 2 l to 4 l per well) showed a significant decrease in gene expression (Figure 2) suggesting that siRNA-mediated reduction in mRNA increases with increasing volumes of transfection agent.
Effect of siRNA concentration on transfection. Increasing amounts of GAPDH siRNA (3, 10, 30, or 100 nM) and negative controls were tested using 3 l transfection reagent to establish the concentration resulting in a >50% reduction in mRNA expression. The most significant siRNA activity was attained using siRNA concentrations between 30 and 100 nM (Figure 3).
Figure 1. Determining Optimal Cell Plating Density. COS-7 cells grown in a 24 well dish were transfected with 10 nM GAPDH siRNA or negative control siRNA 24 hours after plating at the indicated cell densities using 4 l per well siPORT Amine Transfection Agent. Following transfection (48 hr), the cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. Percent gene expression was calculated as a percentage of gene expression compared with the negative control siRNA.
Figure 2. Determining Optimal Amount of Transfection Agent. COS-7 cells plated 24 hr previously at the optimal cell plating density (3.0 x 104 cells/well) in a 24 well plate were transfected with 10 nM GAPDH siRNA or negative control siRNA using 2, 3, or 4 l siPORT Amine Transfection Agent (Ambion)/well. 48 hours following transfection the cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. Percent gene expression was calculated as a percentage of gene expression compared to the negative control siRNA.
Figure 3. Determining Optimal Amount of siRNA. COS-7 cells were plated at the optimized cell plating density (3.0 x104 cells per well) in a 24 well dish. 24 hours after plating, cells were transfected with 100, 30, 10, or 3 nM chemically syntheesized GAPDH siRNA or negative control siRNA using the optimized volume of transfection agent (3 l per well). 48 hours following transfection, the cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. Percent gene expression was calculated as a percentage of gene expression compared to the negative control siRNA.
Important Parameters in siRNA Electroporation Experiments
Not unlike chemical transfection techniques, to ensure maximum delivery of siRNA by electroporation, various parameters need to be optimized for each cell type. Several variables can be tested in order to determine the ideal electroporation conditions. These include field strength, pulse length, number of pulses, type of electroporation buffer, quality of DNA or RNA, temperature, and cell density. Here we focus on the most critical parameters (field strength, pulse length, and number of pulses) to find a set of conditions that maximize the transfection of siRNA and cell viability of PC-12 rat pheochromocytoma cells.
For any new cell type, a series of experiments should be performed to determine optimal electroporation conditions. The voltage applied across the cells is referred to as the field strength (V/cm) and greatly determines overall cell survival and transfection rate. When the field strength of the pulse is high enough, reversible permeation occurs in the cell membrane that allows outside molecules to enter the cell. This event is dependent on several factors including cell diameter, temperature, and the gap width of the cuvette. In general, when determining ideal field strength, optimal transfection efficiency for most cells is often achieved at higher voltages. Also, as a general rule, small cells require higher field strengths than larger cells. But increasing the voltage can also promote cell damage and increase the temperature of the reaction, further reducing cell viability. To compensate for this, low-conductivity electroporation buffers can be used that resist temperature shifts and improve cell survival (Cyto Pulse Low Conductivity Media, Cyto Pulse Sciences, Columbia, MD).
Other key factors for successful electroporation include pulse length and the number of pulses applied. Pulse length is largely dependent on cell diameter. In general, larger cells require longer pulses for successful permeation of the membrane. For most mammalian cell types one pulse is usually sufficient, but multiple pulses may be required to achieve the desired level of silencing with siRNA in some cell types. Because the fine-tuning of electroporation involves varying parameters that can significantly influence cell viability, for these optimization experiments, studies to determine cell viability before and after electroporation events can be crucial.
Transfection efficiency is also affected by the concentration, the purity, and the size of the nucleic acid used. As with chemical methods, transfection efficiency can be modulated by varying the siRNA concentration within a limited concentration range. Experiments with different cell types reveal that the effective concentration of siRNA is somewhere between 100 nM to 1 M in a standard electroporation reaction. siRNA amounts in excess of 2 M may lead to an increase in the transfection efficiency, but may also contribute to cytotoxicity in some cell types. (Experiments detailing these parameters are provided in the article Efficient Delivery of siRNAs to Human Primary Cells.) Although transfection efficiency is affected by the size of the nucleic acid electroporated, we have found that parameters optimized for plasmids can usually be used as starting points for optimizing electroporation of siRNA.
siRNA Experiments Addressing Electroporation Variables
In order to develop optimized conditions for siRNA delivery to PC-12 cells, several electroporation variables were tested. Electroporation experiments were performed using a Cyto Pulse PA-4000S - Advanced PulseAgile Rectangular Wave system (Cyto Pulse Sciences) and Cyto Pulse Low Conductivity electroporation media. PC12 cells were routinely subcultured prior to the day of transfection to ensure healthy cultures. In the following experiment, cells were harvested and resuspended in electroporation media to 6x106 cells/ml. Approximately 0.14 g siRNA was added to each 2 mm gap cuvette containing 100 l of cell suspension for a final siRNA concentration of 100 nM. After briefly mixing, the suspension was electroporated in a standard cuvette holder at the indicated wave forms (Figure 4). Cell viability was determined before and after electroporation using the Guava PCA and ViaCount Assay (Guava Technologies, Hayward, CA). Approximately 48 hr after transfection, total RNA from the cell cultures was extracted using the RNAqueous-4PCR Kit, and quantitated. The expression level of both target and control genes was determined for each sample using Northern analysis (NorthernMax-Gly Kit). Bands were quantitated by densitometry.
Effect of varying number of pulses on transfection of siRNA. Varying the number of pulses was overall the most influential parameter in the experiment. For PC12 cells, one pulse was sufficient to achieve optimal siRNA delivery (Figure 4). High levels of target mRNA reduction was observed for some cells pulsed three and five times. However, the data was variable and showed marked effects on cell viability.
Effect of pulse length and field strength on transfection of siRNA. To optimize the duration of the pulse we tested pulse lengths of 100 s, 250 s, and 400 s (Figure 4). Optimal siRNA activity without significant loss of cell viability was observed for PC-12 cells between 250 and 400 s pulses. Increased gene silencing correlates with increased field strength from 300 to 500 V/cm. The cell mortality rate also increased with increased field strength with the most significant change in viability at 500 V/cm.
Figure 4. Effects of Electroporation Field Strength and Pulse Length. PC-12 rat pheochromocytoma cells were electroporated using a Cyto Pulse PA-4000S - Advanced PulseAgile Rectangular Wave system and Cyto Pulse Low Conductivity electroporation media. Varying wave forms were tested to determine the optimal conditions for the delivery of 140 ng GAPDH siRNA. 48 hours post-transfection, cells were harvested and analyzed by Northern blot analysis and bands were quantitated by densitometry. Percent gene expression was calculated as a percent of non-electroporated controls. Duplicates were performed for each sample. Cell viability was carried out using the using the Guava PCA and ViaCount Assay. Cell viability was calculated as a percent of viable cells before electroporation and two hours post-electroporation.
A comprehensive set of data was collected from different transfection conditions using two different methods of delivery, chemical transfection and electroporation. It is apparent that there is no single transfection parameter that by itself ensures efficient siRNA uptake by cells in culture. Increased uptake of siRNA into viable cells is achieved by systematically addressing each of several critical variables. For chemically mediated methods those variables are cell density and reagent concentration. For electroporation, a set of data collected at over 25 different conditions shows the dependence of delivery and viability on 3 independent parameters: field strength, pulse length and number of pulses. These data should provide a tool to develop insight into siRNA transfection mechanisms and a guide to optimize both chemical transfection and electroporation protocols. l
Note that it is common to see relative variations in data when conducting tissue culture experiments. Reproducibility can be achieved by rigorously following protocols.
siPORT Amine is manufactured for Ambion by Mirus.
TaqMan is a registered trademark of Applied Biosystems.