General: Definitions and Techniques
FAQ: What is transfection and what are the commonly used methods?
HelpCorner: Transfection is the delivery of DNA, RNA, proteins, and macromelecules into eukaryotic cells. The goals for transfection include the study of gene function and regulation as well as protein expression and function. It is also used for gene silencing and protein production. According to the application, the information can be either transiently (transient transfection) or permanently expressed (stable transfection). Based on the strategies, the transfection methods can be divided into three groups:
- methods that rely on carrier molecules,
- methods that deliver nucleic acids directly to the cytoplasm,
- methods that employ viral vectors.
Carrier molecules: Six different carriers are commonly used:
- DEAE Dextran, a polymeric cation which associates tightly with the negatively charged DNA and carries it into the cell
- A calcium phosphate-DNA coprecipitate which is taken up by cells in culture
- Ionic interaction of DNA and liposomes to form a complex (e.g., DOTAP and DOSPER)
- A complex mixture of a different components or multicomponent (e.g., FuGENE® HD Transfection Reagent)
- Receptor-mediated transfection
- Particle-based transfection (highly charged polymeric substances)
Direct delivery to the cytoplasm: The two main techniques are:
- Electroporation: a high concentration of cells is briefly exposed to a high-voltage electric field in the presence of the DNA to be transfected.
- Particle-mediated gene transfer, also known as the biolistic or gene gun method, employs microscopic gold particles as delivery vehicles to introduce DNA into targeted cells.
Virus vectors: The gene of interest is cloned into the viral genome in vitro. Viral particles are assembled in special cells prior to infection of the target cells.
FAQ: What does “reverse transfection” mean?
HelpCorner: The term “reverse transfection” comes from the invention and development of a microarray-driven gene expression system by Junald Ziauddin and David M. Sabatini in 2001. As DNAs are printed on a glass slide for the transfection process to occur prior to the addition of adherent cells, the order of addition of DNA and adherent cells reverses that for conventional transfection. It is usually used for high-throughput protocols.
Transfection Reagents and Applications
FAQ: Which product do you recommend for transfection of siRNA?
HelpCorner: We recommend using X-tremeGENE siRNA Transfection Reagent from Roche Applied Science. It is an optimized lipid-based transfection reagent that forms a complex with short interfering RNA (siRNA) and with mixtures of siRNA and plasmid DNA. Another powerful transfection agent, the FuGENE® HD Transfection Reagent has also been successfully used for siRNA Knock-Down assays (Biochemica No.1, 2008, page 21-24).
FAQ: What should I pay attention to when transfection is either a downstream or an upstream assay?
HelpCorner: Transfection as a downstream assay is used to introduce new information into living cells. To fully exploit this important technique, the quality and the purity of the material to be inserted must be high and carefully checked. The downstream assay of a transfection experiment will define the level of expression you want to achieve. If you are using microscopic techniques to study the expression of your transfected gene, you may not want a high transfection efficiency; rather, you may want to keep the morphology of the cells unchanged (low cytotoxicity). If the purpose of your assay is the production of large amounts of a protein, you will want to achieve a very high transfection efficiency to have a high yield.
Analyzing purity of DNA via optical density (OD) measurement
An A260/A280<1.8 indicates that the preparation is contaminated with proteins and aromatic substances. An A260/A280>2 indicates a possible contamination with RNA. The OD gives no information about the size of the DNA.
FAQ: Is it possible to directly use PCR constructs for a transfection?
HelpCorner: Yes, it is possible to directly use PCR constructs and check the expression of the protein of interest. FuGENE® HD Transfection Reagent was used to deliver PCR fragments to HeLa cells (Biochemica No. 4, 2007, page 22-25).
FAQ: Can FuGENE® Transfection Reagents be used to transfect stem cells?
HelpCorner: Yes, you can use FuGENE® HD Transfection Reagent to transfect stem cells as shown in Biochemica (No. 4, 2006, page 19-21). You can find all information concerning the cells transfected with Roche Applied Science products under www.powerful-transfection.com.
FAQ: Which product would you recommend for transfecting large plasmids?
HelpCorner: It is possible to transfect large plasmids using FuGENE® HD Transfection Reagent. You can find some examples in Biochemica (e.g., Biochemica No. 2, 2008).
FAQ: Do you have a standard protocol for transfection?
HelpCorner: No single transfection protocol can be applied to all types of cells and experiments. Varying degrees of optimization may be required to obtain maximal transfection efficiency.
FAQ: Should I use antibiotics during the transfection protocol?
HelpCorner: We do not recommend the use of antibiotics during the transfection procedure.
Transfection procedure using FuGENE® HD Transfection Reagent:
- Allow FuGENE® HD Transfection Reagent, DNA, and diluent to adjust to +15 to +25°C. Vortex for one second or invert the FuGENE® HD Transfection Reagent vial to mix.
- Dilute DNA with appropriate diluent, for example, Opti-MEM I Reduced Serum Medium, serum-free medium (without antibiotics or fungicides), or sterile water to a concentration of 2 µg plasmid DNA/100 µl Opti-MEM (0.02 µg/µl).
- Place 100 µl diluent, containing 2 µg DNA, into each of six sterile tubes labeled 3:2, 4:2, 5:2, 6:2, 7:2, and 8:2.
Recommendation: Use sterile polystyrene tubes or round-bottom, 96-well plates to form the transfection complex.
- Form the transfection complex by adding FuGENE® HD Transfection Reagent to tubes containing diluted DNA:
Pipet the FuGENE® HD Transfection Reagent (3, 4, 5, 6, 7, or 8 µl) directly into the medium containing the diluted DNA without allowing contact with the walls of the plastic tubes.
- Mix and incubate the transfection complex:
Vigorously tap the tube or vortex for 1–2 seconds to mix the contents. If using a 96-well plate, place the plate on a rotating shaker for 5–10 seconds. Incubate the transfection reagent: DNA complex for 15 minutes at room temperature. For some ratios and cell types incubation is not necessary for optimal complex formation, while a longer incubation time is better for other cell types. Determine this for your particular cell line and the ratio you use.
- Add the transfection complex to cells:
Remove culture vessel from the incubator. Removal of growth medium is not necessary. Add the transfection complex to the cells in a drop-wise manner or add below the surface of the medium. Swirl the wells or flasks to ensure distribution over the entire plate surface. Use of a rotating platform shaker for 30 seconds at low speed provides adequate mixing for 96-well plates. Once the FuGENE® HD Transfection Reagent: DNA complex has been added to the cells, there is no need to remove and replace with fresh medium (as is necessary with some other transfection reagents).
- Incubate cells and assay the results:
Following transfection, incubate the cells for 18–72 hours prior to measuring protein expression. The length of incubation depends upon the transfected vector construct, the cell type being transfected, the cell medium, cell density, and the type of protein being expressed. Following this incubation period, measure protein expression using an assay that is appropriate for your system.
Optimization and Troubleshooting
FAQ: Which controls should I include to check the conditions used for my protocol?
HelpCorner: If you are working with vector plasmids, you should always include a control vector which does not contain the DNA of interest. In order to check the transfection efficiency, we recommend using immunocytochemistry, FACS or biochemistry (e.g., western blot or immunoprecipitation) to assay the expression of the protein of interest. If you want to check carefully the level of expression, you can also perform a real-time PCR assay. We strongly recommend verifying the viability of your cells using our new xCELLigence System. Transfection is easily done using E-Plates 16 or 96, containing 16 and 96 wells, respectively.
FAQ: Which parameters can influence the efficiency of a Transfection Reagent?
HelpCorner: The critical variables for transfection are:
- Condition of cell culture:
a. Aseptic techniques/contamination (e.g., mycoplasma: slower growth)
b. Culture vessels (e.g., substrates: a change of the plastic quality may influence the growth of your cells)
c. Media and supplements (e.g., transfection media, with or without serum)
d. Subculture (e.g., passage number)
e. Cell density and quantitation
f. Cell viability, cell proliferation and cytotoxicity
- Vector construct or gene of interest
a. Integrity (e.g., wrong storage)
b. Preparation (e.g., dilution solution) and purification
- Transfection method/technique
a. Type of transfection reagent
b. Amount of transfection reagent
c. Ratio: plasmid/transfection reagent
d. Amount of complex added
e. Timing (e.g., duration of the incubation)
FAQ: How can I optimize the transfection efficiency with FuGENE® HD Transfection Reagent?
HelpCorner: In order to optimize your protocol, we recommend testing three parameters in a simple optimization experiment. Six different complexes are made using FuGENE® HD Transfection Reagent, and different amounts of the complexes are then added to the wells of a 96-well plate at three different time points (Table 1).
- Ziauddin, J. & Sabatini, D.M. Microarrays of cells expressing defined cDNAs. Nature 411, 107-110 (2001).
- Tools for transfection ()
- FuGENE® HD Transfection Reagent ()
- Tips and tricks on how to improve transfection experiments ()
- Transfection Control Experiment (
- Protocol for optimizing transfection of adherent cell lines ()
- Identifying and Solving Transfection Problems ()
- Culture and monitoring of animal cells - Basic techniques ()
- Lab FAQs, 3rd Edition ()
- Apoptosis and Cell Proliferation Manual, 3rd edition ().
This article was originally published in Biochemica 3/2009, pages 23-25. ©Springer Medizin Verlag 2009