Low transfection efficiency is the most frequent cause of unsuccessful gene silencing experiments. We have found that by optimizing cell density, transfection agent amount, and siRNA concentration, high 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 can be transiently transfected into mammalian cells using commonly available transfection reagents. However, obtaining high efficiency transfection of siRNA is not trivial and may limit the utility of RNAi in some cell types.
Recent publications have described the use of cationic liposomal and polyamine based agents to facilitate delivery of siRNA into cultured mammalian cells (Byrom et al., 2002; Demeterco et al., 2002; Harborth et al., 2001). To achieve maximum effectiveness of exogenously introduced siRNAs, transfection optimization experiments are required. Failure to optimize some critical transfection parameters, such as cell culture conditions, the choice, amount, and use of transfection agents, and quantity and quality of siRNAs can render siRNA effects undetectable in tissue culture. Once an optimized protocol is developed for a particular cell type, siRNAs can be easily screened with a high degree of reproducibility.
This article summarizes optimization experiments for m 4505 siPORT Lipid Transfection Agent 1 ml aximizing the performance of siRNA in tissue culture. We discuss the use of chemical transfection agents and offer suggestions on how to optimize critical transfection parameters.
Important Parameters in siRNA Transfection Experiments
Cells vary greatly with respect to their capacity to be transfected. Hence, different transfection reagents and cell culture conditions need to be tested for each cell type. What works for one cell type won't necessarily work for another. Some cells perform best when transfected in serum supplemented media while others require serum free conditions for maximal transfection efficiency. Healthy cells transfect better than poorly maintained cells. Routinely subculturing cells before they become overcrowded or unhealthy will minimize instability in continuous cell lines from experiment to experiment. Since cells may gradually change in culture, using cells within a defined passage number and maintaining strict protocols, including parameters for intervals between plating and transfecting cells, will improve experimental reproducibility.
Cell confluency. An important factor for obtaining a high transfection efficiency is cell density at the time of transfection. For most adherent cells, the optimal confluency for transfection is 30-70%. Suboptimal cell density (too low or too high) can result in poor uptake of the siRNA:transfection agent complexes and insufficient silencing of the gene of interest.
Choice of transfection agent. The overall tran sfection efficiency and degree of gene silencing is dependent on the nature of the transfection agent/siRNA complex. The appropriate transfection agent varies with cell type. Ambion recommends using an agent that has been validated specifically for siRNA transfection (see Ambion's recommendations). For special cell types that are difficult to transfect, different types of transfection reagents (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 siRNA significantly influences RNAi experiments. siRNA should be free of reagents carried over from synthesis including salts, proteins, and ethanol. Also, the presence of dsRNA larger than approximately 30 bp has been shown to activate the nonspecific interferon response (Stark et al., 1998). The optimal concentration of siRNA is influenced by several factors including properties of the target gene, cell type, and target. Too much siRNA may result in cytotoxicity. Conversely, if too little siRNA is transfected, gene target knock down may be undetectable.
Experiments Addressing Transfection Variables
Several experiments were designed to test transfection conditions. For these experiments, cells were plated into a 24 well dish one day before transfection, unless otherwise noted. Transfections were perfor med using siPORT Amine Transfection Agent according to protocol with suggested concentrations of chemically synthesized siRNA in tissue culture media. 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 either Northern analysis (NorthernMax-Gly Kit) or real-time RT-PCR with gene specific primers and TaqMan probes. For real-time assays, transfections were performed in triplicate and all samples were normalized to18S rRNA levels.
Effect of serum on transfection. Five cell lines were chosen to assess the effect of serum on transfection. Cells were transfected with either GAPDH siRNA or with a negative control (scrambled) siRNA using two different types of transfection agents (siPORT Amine and siPORT Lipid) in either serum free or serum supplemented media (10% serum). The ability of GAPDH siRNA to reduce GAPDH mRNA levels was evaluated by Northern blot analysis. We found that individual cell types responded differently to serum free versus serum supplemented conditions, depending on the specific transfection agent used (Figure 1). When transfected under optimal conditions, GAPDH mRNA expression levels were effectively reduced by the corresponding siRNA, but not by the negative control siRNA. In contrast, little to no mRNA reduction was observed in several of the cell types when suboptimal media conditions were used. For the remainder of the study, COS-7 cells were transfected in serum supplemented media using siPORT Amine Agent.
Figure 1. Effect of Serum on siRNA Transfection. The indicated cells types were transfected with GAPDH siRNA or negative control siRNA using either siPORT Amine or siPORT Lipid Transfection Agent (Ambion) in cell culture media supplemented with (+) or without () serum (10%) for 4 hr. 48 hours after transfection GAPDH mRNA levels were assessed by Northern analysis.
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 2). 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 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 (Figure 2). To minimize decreased cell growth associated with very low plating densities, an optimal cell plating density of 3 x 104 cells per well wa s used for the remainder of the study.
Figure 2. 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.
Effect of varying amounts of transfection agent on cytotoxicity and siRNA activity. To study the effects of increasing amounts of transfection agent on siRNA activity, COS-7 cells (3 x 104 cells per well) were plated into a 24 well dish 24 hours prior to transfection. The relative cytotoxicity of transfection agent/negative control GAPDH siRNA (10 nM) complex at increasing volumes of transfection agent was assessed by Trypan Blue staining of cells 48 hours post transfection. To characterize the effects of transfection agent levels on siRNA-mediated silencing, cells were transfected using different volumes of transfection agent as shown in Figure 3. mRNA expression was evaluated by real-time RT-PCR 48 hr post transfection. The data indicate that cell survival decreased significantly when cells were exposed to transfection agent/negative control GAPDH siRNA complexes at volumes above 4 l per well (Figure 3). Increasing amounts of transfection agent (from 2 l to 4 l per well) showed a dramatic decrease in gene expression (Figure 4) suggesting t hat siRNA-mediated reduction in mRNA increases with increasing volumes of transfection agent. A midpoint volume of 3 l per well was used for all remaining experiments.
Figure 3. Cytotoxicity of Transfection Agent/siRNA complexes. COS-7 cells were plated into wells of a 24 well plate at 3.0 x 104 cells per well approximately 24 hr prior to transfection. Complexes containing negative control siRNA (10 nM) and various amounts of siPORT Amine Transfection Agent were added to the wells. After transfection (48 hr), cells were washed, trypsinized, and stained with 10% Trypan Blue. Cells were subsequently counted and percent cell survival was calculated as: (Total number of cells - Number of stained cells) / Total number of cells x 100.
Figure 4. Determination of Optimal Amount of Transfection Agent. 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 optimized cell plating density (3.0 x 104 cells per well) using 2, 3, or 4 l siPORT Amine Transfection Agent 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.
Effect of siRNA Concentration on Transfection. We tested several siRNA concentrations to establish the amount required to achieve >50% mRNA reduction. Increasing amounts of GAPDH siRNA (3, 10, 30, and 100 nM) and negative controls were tested with t he optimized amount of cells (3 x 104 cells/well) and 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 5). We were able to achieve approximately 75%-90% reduction in GAPDH mRNA levels over control with no apparent cytotoxicity.
Figure 5. Determination
of 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.
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.
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3. Harborth J, Elbashir SM, Bechert K, Tuschl T, Weber K (2001) Identification of essential genes in cultured mammalian cells using small interfering RNAs. J Cell Science 114: 45574565.
4. Stark, G.R., Kerr, I.M., Williams, B.R., Silverman, R.H. and Schreiber, R.D. (1998). How Cells Respond to Interferons. Annu. Rev. Biochem 67: 227264.
For prices and availability, please contact our Customer Service Department. Cat# Product Name Size 4502 siPORT Amine Transfection Agent 0.4 ml 4503 siPORT Amine Transfection Agent 1 ml 4504 siPORT Lipid Transfection Agent 0.4 ml