You will need to optimize specific transfection conditions to achieve the desired transfection efficiencies. Important parameters to consider are the charge ratio of cationic lipid transfection reagent to DNA, amount of transfected nucleic acid, length of time cells are exposed to the transfection reagent and presence or absence of serum. Reporter genes are useful to determine optimal conditions. The transfection efficiency achieved using any transfection reagents varies depending on the cell type being transfected and transfection conditions used.
Charge Ratio of Cationic Transfection Reagent to Nucleic Acid
The amount of positive charge contributed by the cationic lipid component of the transfection reagent should equal or exceed the amount of negative charge contributed by the phosphates on the DNA or RNA backbone, resulting in a net neutral or positive charge on the multilamellar vesicles associating with the DNA or RNA. Optimization of the lipid:DNA ratio is particularly important if the culture medium contains serum (Chan et al. 2014; Tranchant et al. 2004).
Nucleic Acid Amount
The optimal amount of DNA or RNA will vary depending on the type of nucleic acid, number of cells, culture dish size and target cell line used. For example, COS-7 cells are optimally transfected with 100ng of pGL4.13[luc2/SV40] Vector (Cat.# E6681) using ViaFect™ Reagent at a 4:1 ratio in a 96-well plate. In contrast, the same cells are optimally transfected with 50ng of DNA using the FuGENE® HD Transfection Reagent at a 3:1 ratio in the same well size. For other cell lines, we suggest testing the nucleic acid amounts given in Table 2.
Table 2. Suggested Nucleic Amounts to Use for Optimization.
||Nucleic Acid Amount to Test
||Reagent:Nucleic Acid Ratios to Test
||Culture Dish Size
||6:1, 5:1, 4:1, 3:1, 2.5:1 and 2:1
|FuGENE® 6 Transfection Reagent
||4:1, 3.5:1, 3:1, 2.5:1, 2:1 and 1.5:1
|FuGENE® HD Transfection Reagent
||4:1, 3.5:1, 3:1, 2.5:1, 2:1 and 1.5:1
|FuGENE® 4K Transfection Reagent
|FuGENE® SI Transfection Reagent
Significantly increasing the quantity of transfected nucleic may not yield better results. In fact, if initial transfection results are satisfactory, a reduced DNA or RNA quantity can be tested (while keeping the optimal reagent:nucleic ratio constant). Often a range of DNA or RNA concentrations is suitable for transfection. However, if the nucleic acid concentration is below or above this range, transfection efficiencies will decrease. If there is too little DNA or RNA, the experimental response may not be present. If there is too much nucleic, the excess can be toxic to cells. Calibrate the system using a test plasmid with reporter gene function.
Traditionally, transfection reagents must remain in contact with cells for a defined period of time, after which additional medium is added or the medium is replaced to help minimize toxic effects of the reagent. The optimal transfection time depends on the cell line, transfection reagent and nucleic acid used. For the ViaFect™ Transfection Reagent and the FuGENE® HD Transfection Reagent, which are two of the more gentle methods of DNA transfection into cells, there is no need to add more medium or replace the medium after transfection.
For initial tests with liposomal reagents that require adding or replacing the medium, use a 1-hour transfection interval, and test transfection times of 30 minutes to 4 hours or even overnight, depending on the reagent used. Monitor cell morphology during the transfection interval, particularly when cells are maintained in serum-free medium, because some cell lines lose viability under these conditions.
Transfection protocols often require serum-free conditions for optimal performance because serum can interfere with many commercially available transfection reagents (Chan et al. 2014). The ViaFect™, FuGENE® HD and
Transfection Reagents can be used in transfection protocols in the presence of serum, ideal for transfecting cell types (e.g., primary cells) or applications that require continuous exposure to serum.. Note that the best results are obtained when variability is minimized among different lots of serum.
Co-Transfection and Dual-Reporter Assays
Although many experiments use a single reporter gene, a dual-reporter system has distinct advantages. A second reporter gene can normalize expression for transfection efficiency and cell number. Small perturbations in growth conditions for transfected cells can dramatically affect gene expression. A second reporter helps to determine if the effects are due to the treatment of the cells or a response from the experimental reporter (Chu and Rana 2008; Zhou et al. 2016).
The Nano-Glo® Dual-Luciferase® Reporter Assay System (NanoDLR) (Cat.# N1610) is an efficient means of quantitating luminescent signals from two reporter genes in the same sample. In this system, firefly (Photinus pyralis) and NanoLuc® luciferase activities are measured sequentially from a single sample in a homogeneous format. In the NanoDLR™ System, both reporters yield linear assay responses (with respect to the amount of enzyme) and exhibit no endogenous activity in experimental host cells. In addition, the extended half-life of the reporter signals are ideal for use with multiwell assay formats.
The various Promega Renilla luciferase vectors can be used as control vectors when co-transfected with a firefly luciferase vector into which the promoter of interest is cloned. Alternatively, the firefly vector may be used as the control vector and the NanoLuc® luciferase vector as the experimental construct. In a co-transfection experiment, note that trans effects between promoters on co-transfected plasmids can potentially affect reporter gene expression. This is primarily of concern when either the control or experimental reporter vector, or both, contain very strong promoter/enhancer elements. The occurrence and magnitude of such effects will depend on several factors: 1) the combination and activities of genetic regulatory elements present on the co-transfected vectors; 2) the amount and relative ratio of experimental vector to control vector introduced into cells; and 3) the cell type transfected.
To help ensure independent genetic expression of experimental and control reporter genes, preliminary co-transfection experiments should be performed to optimize both the vector DNA amount and ratio of co-reporter vectors. Because NanoLuc® luciferase produces an extremely bright signal, it is possible to use very small quantities of these vectors to provide low-level, constitutive co-expression of NanoLuc® luciferase activity. This means that the ratio of firefly and NanoLuc® luciferase vectors to test vector can range from 1:1 to 10,000:1 (or greater) to determine the optimal expression. The key to a dual-reporter system is to maximize expression of the experimental reporter while minimizing that of the control reporter. However, the expression level of the control reporter should be three standard deviations above background to be significant.
The strength of the promoter in your cell system is an important consideration. A more moderately expressing promoter like thymidine kinase [TK; e.g., pNL1.1.TK(Nluc/TK) ] Vector (Cat.# N1501)] may be preferable to SV40 or CMV. Stronger promoters may exhibit more trans effects, cross-talk or regulatory problems. However, adjusting the ratio of experimental vector to control vector (e.g., using 100:1 or 200:1) may eliminate some of these issues.
When transfecting nucleic acids into cells, there are controls that can help assess the transfection. These recommended controls include:
- Cells control: Medium alone (e.g., 5µl of medium)
- DNA/RNA control: DNA or RNA without transfection reagent (e.g., 100ng DNA in a 5µl volume)
- Reagent control: Transfection reagent without DNA/RNA (e.g., 0.4µl of reagent in a 5µl volume)
If any of these controls do not provide the expected result, consider repeating the transfection with new cells, medium, transfection reagent or any combination of these components.