Transfection Protocols & Applications

Transient transfection

When cells are transiently transfected with plasmids, the DNA is introduced into the nucleus of the cell, but does not integrate into the chromosome. This means that many copies of the gene of interest are present, leading to high levels of expressed protein. Transcription of the transfected gene can be analyzed within 24–96 hours after introduction of the DNA depending on the construct used. Transient transfection is most efficient when supercoiled plasmid DNA is used. siRNAs miRNAs and mRNAs can be used for transient transfection and are effective in the cytoplasm, without the need to be transferred to the nucleus.

Stable transfection

With stable or permanent transfection, the transfected DNA is either integrated into the chromosomal DNA or maintained as an episome. Stable integration of plasmid DNA into the genome is a rare event. Stably transfected cells can be selected by co-transfection of a second plasmid carrying an antibiotic-resistance gene or by providing a resistance gene on the same vector as the gene of interest. siRNA and miRNA can only be stably transfected when they are delivered as short hairpin transcripts made from a selectable DNA vector. However, RNA molecules per se cannot be used for stable transfection.

Although linear DNA results in lower DNA uptake by the cells relative to supercoiled DNA, it yields optimal integration of DNA into the host genome. Cells which have successfully integrated the DNA of interest or have maintained episomal plasmid DNA can be distinguished by using selectable markers. Frequently used selectable markers are the genes encoding aminoglycoside phosphotransferase (APH; neoR gene) or hygromycin B phosphotransferase (HPH). Other selectable markers are the genes encoding adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hymidine kinase (TK), or xanthine-guanine phosphoribosyl transferase (XGPRT; gpt gene).

Fast-forward and reverse transfection

In fast-forward transfection, plating and transfection of cells are performed on the same day. DNA or siRNA is diluted in culture medium without serum. Transfection reagent is added to the diluted nucleic acid (NA) to produce reagent–NA complexes. The cells are seeded and then complexes are added directly to the freshly seeded cells. Whereas, in a traditional transfection, cells are plated 24 hours prior to transfection. Cells are seeded in culture medium containing serum and antibiotics the day before transfection and incubated under normal growth conditions. The next day, DNA or siRNA is diluted in culture medium without serum. Transfection reagent is added directly to the diluted DNA or siRNA to produce reagent–NA complexes. During complex formation, the medium on the cells is changed, before the complexes are added to the cells (see figure Fast forward and reverse transfection).

In standard or fast-forward transfection, cells are added to plate wells first, followed by transfection complexes. In reverse-transfection, nucleic acid is added to plate wells, followed by transfection reagent. Cells are added after complex formation in the wells, hence the term “reverse transfection” (see figure Steps in fast-forward and reverse transfection).

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General guidelines for successful transfection

Successful transfection is influenced by many factors. The health and viability of the cell line, quality of the nucleic acid used, transfection reagent, duration of transfection, and the presence or absence of serum can all play a part. 

Cells 

Cells should be grown in an appropriate medium, supplemented with serum or growth factors, as required for the viability of that cell line. Do not use cells and media that you know are contaminated (e.g., with yeast or mycoplasma). If there is any doubt, reseed cells from a frozen, uncontaminated stock. Always ensure the medium is fresh if any components are unstable since missing key components may harm cell growth. Keep the incubation conditions constant at 37°C, CO₂ at the correct level (usually 5–10%) and 100% relative humidity.

Every cell line has optimal culture conditions. Refer to the American Type Culture Collection [ATCC] web site.  

Serum

Some transfection protocols require serum-free conditions for optimal performance, since serum can interfere with many commercially available transfection reagents. This should be checked for your protocol.

Confluency

As a guide, cells should be transfected at 40–80% confluency. Too few cells will cause the culture to grow poorly without cell-to-cell contact. Too many cells results in contact inhibition, making cells resistant to uptake of foreign nucleic acid. Actively dividing cells will yield best results.

Passages 

Keep the number of passages low (<50). In addition, the number of passages for cells used in a set of experiments should be consistent. Always take care to make sure that the cell cultures to be transfected are actively dividing, and are at least 90% viable, prior to transfecting. Cell characteristics can change over time with immortalized cell lines, and cells may not respond to the same transfection conditions after repeated passages, resulting in poor expression.

Quantity of nucleic acids

The optimal amount of nucleic acid varies widely, depending on the type of nucleic acid, number of cells, size of culture dish/plate, and the cell line used.

Increasing the quantity of transfected nucleic acid significantly may not yield better results. In fact, if initial transfection results are satisfactory, a reduced nucleic acid quantity should be tested (keeping the optimal reagent: nucleic acid ratio constant).

In some cases, a range of nucleic acid concentration may be suitable for transfection; although efficiency will decrease, sometimes markedly, outside of this range.

Too little nucleic acid may result in the experimental response not being present. Conversely, too much nucleic acid can prove toxic to cells.

Amount of transfection reagent

This is dependent upon the transfection reagent being used and should be optimized carefully.

Transfection complex formation and complex incubation time

Many chemical transfection reagents have an ideal time window, in which a transfection complex of optimal diameter is formed. This is typically between 5 and 30 minutes, depending upon the nature of the reagent. Refer to the reagent manufacturer’s recommendations.

In general, transfection reagents need to be in contact with cells for a period of time before 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.

In some instances, plating cells onto wells or plates containing transfection complexes may result in increased transfection efficiency, compared to the traditional approach of adding transfection complexes to an established culture. An additional benefit to such reverse transfection protocols is that seeding and transfecting cells on the same day shortens the experimental timeline by a full day.

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Guidelines for transfection of DNA

Considerations for plasmid DNA transfection

DNA quality strongly influences the results of transfection experiments. The best results are achieved when plasmid DNA of the highest purity is used for transfection.

Endotoxins, also known as lipopolysaccharides (LPS), are cell membrane components of Gram-negative bacteria (e.g., E. coli) that are released during the lysis step of plasmid preparation and are often copurified with plasmid DNA. The presence of endotoxins in plasmid DNA can result in sharply reduced transfection efficiencies, particularly in primary cells and sensitive cell lines. For transfection of endotoxin-sensitive cells (e.g., primary cells, suspension cells, and hematopoietic cells) or for highest transfection efficiencies and lowest cytotoxicity, we recommend using DNA purified using a commercially available kit specifically designed for endotoxin removal. Such kits help to ensure optimal transfection results.

The configuration of a DNA molecule used for transfection influences the efficiency of transfection. Transient transfection is most efficient with supercoiled plasmid DNA. In stable transfection, linear DNA yields optimal integration into a host cell’s genome although uptake into cells is lower compared with plasmid DNA.

If the gene product of DNA transfected into a cell is toxic to the cell, its expression will lead to cell mortality. In such cases, it may be necessary to use either a weak or an inducible promoter to limit the damage caused to cells due to expression of a toxic gene product.

Choice of transfection technology

Several methods for transfection of nucleic acids have been developed using DEAE-dextran, calcium phosphate, electroporation, liposomes, non-liposomal lipids, and activated dendrimers. The choice of transfection technology can strongly influence transfection efficiency. Ideally, transfection should be fast and easy to perform, give high efficiencies and reproducible results, and cause minimal cytotoxicity.

The importance of optimization

For the best results and the most efficient transfection, an optimal ratio of DNA to transfection reagent must be determined for each cell line–plasmid combination. Therefore, it is vital that optimization trials are carried out for each new cell line–plasmid combination used.

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Guidelines for transfection of RNA

The transfection of RNA has become extremely important with the discovery of RNA interference (RNAi)-mediated gene silencing using short interfering RNA (siRNA).

Considerations for transfected RNA

Various forms of RNA, such as mRNA, in vitro transcribed RNA, viral RNA, RNA oligos, siRNA, and ribozymes, can be used for transfection. If using mRNA, the presence or absence of features such as a 5' cap, internal ribosomal entry site, or poly-A tail can have a significant effect on efficiency of transfection.

Optimal transfection results are achieved when RNA of the highest purity, free of contaminating DNA and proteins, is used for transfection. It should be considered that if the expressed gene product is toxic to the cell, overexpression may lead to cell death.

For RNAi experiments, high-purity, ready-to-use siRNA is available from several commercial suppliers. It is essential to use the correct sequence to achieve efficient silencing. Note that silencing the expression of essential genes may lead to cell death. For more information, see Guidelines for transfection of siRNA.

Avoiding contamination of RNA

No currently available purification method can guarantee that RNA is completely free of DNA, even when no DNA is visible on an agarose gel. For RNA transfection, treatment of the purified RNA with RNase-free DNase or comparable methods is recommended.

Ribonucleases (RNases) are very stable and active enzymes that do not generally require cofactors to function. Since RNases are difficult to inactivate and minute amounts are sufficient to destroy RNA, plasticware, glassware, or solutions should only be used after eliminating possible RNase contamination. Great care should be taken to avoid inadvertently introducing RNases during the transfection procedure. In order to create and maintain an RNase-free environment when working with RNA, proper microbiological, aseptic technique should be followed, and the use of sterile, disposable plastic tubes is recommended.

The importance of optimization

The following factors must be optimized for the best results in RNA transfection: 

• Cell density at the time of transfection
• The ratio of RNA to transfection reagent
• The time of incubation with the transfection complexes

We recommend careful optimization of these parameters for every cell type and RNA combination used. Once optimized, these parameters should be kept constant in all future experiments with each particular combination.

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Guidelines for transfection of siRNA

The application of RNA interference (RNAi) to mammalian cells has revolutionized the field of functional genomics. The ability to simply, effectively, and specifically downregulate the expression of genes in mammalian cells holds enormous scientific, commercial, and therapeutic potential. Efficient transfection of siRNA is critical for effective gene silencing.

Off-target effects in RNAi experiments

Studies have indicated that transfection of siRNA can result in off-target effects, in which siRNAs affect the expression of nonhomologous or partially homologous gene targets. Off-target effects can include mRNA degradation, inhibition of translation, or induction of an interferon response (5–8). The mechanisms of off-target effects are not fully understood. They may be caused by siRNA targeting mRNA with close homology to the target mRNA, by siRNAs functioning like miRNAs, or by a cellular response to siRNA toxicity. In addition, some researchers have observed an siRNA-mediated interferon response.

Research suggests that off-target effects, which may produce misleading results in RNAi experiments, can be largely avoided by using low siRNA concentrations (9, 10).

Optimizing siRNA transfection

Calculating concentrations of siRNA

Approximate values for a double-stranded, 21 nt siRNA molecule:

• 20 µM siRNA is equivalent to approximately 0.25 µg/µl
• The molecular weight of a 21 nt siRNA is approximately 13–15 µg/nmol

To achieve the best results in siRNA transfection of adherent cells, we recommend optimizing the following parameters.

Amount of siRNA

The amount of siRNA used is critical for efficient transfection and gene silencing. The ratio of transfection reagent to siRNA should be optimized for every new cell type and siRNA combination used.

Cell density at transfection

The optimal cell confluency for transfection should be determined for every new cell type to be transfected and kept constant in future experiments. This is achieved by counting cells before seeding and, in the case of using a traditional protocol, by keeping the interval between seeding and transfection constant. This ensures that the cell density is not too high and that the cells are in optimal physiological condition at transfection.

A guide to the number of cells to seed for different formats is shown in the tables Typical number of adherent cells to seed, Typical number of suspension or macrophage cells to seed, and Typical number of primary cells to seed.

Transfection in multiwell plates — preparing a master mix

If you are performing transfection in multiwell plates, prepare a master mix of transfection complexes or of transfection reagent and culture medium (depending on the protocol) for distribution into plate wells.

• Calculate the required volumes of each component and the total volume before you prepare the master mix.
• Prepare 10% more master mix than is required to allow for pipetting errors (i.e., for a 48-well plate, prepare enough master mix for 53 wells).
• Use a repeat pipet to distribute the master mix.

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Performing appropriate RNAi control experiments

It is important to perform suitable control experiments so that results can be correctly interpreted. We recommend the following control experiments.

Positive control siRNA

This is an siRNA that is known to provide high knockdown of its target gene. A positive control is used to establish that the experimental set up for transfection and knockdown analysis is working optimally. An siRNA that knocks down a gene resulting in the phenotypic effect under study may also be used as a positive control to ensure that the phenotypic assay is working optimally. A positive control siRNA should be transfected in every RNAi experiment.

Negative control siRNA

A negative control siRNA should be a nonsilencing siRNA with no homology to any known mammalian gene. Transfection of negative control siRNA is used to determine whether changes in phenotype or gene expression are nonspecific. A negative control siRNA should be transfected in every RNAi experiment.  

Transfection control siRNA

This control is used to measure the transfection efficiency. Transfection efficiency can be measured in several ways, for example by fluorescence microscopy after transfection of a fluorescently labeled siRNA or by observation of the level of cell death after transfection of siRNA that targets essential cell survival genes. siRNA transfection efficiency should be as high as possible. This control should be performed for optimization, for example, when establishing RNAi in a new cell line.

Mock transfection control

Mock-transfected cells go through the transfection process without addition of siRNA (i.e., cells are treated with transfection reagent only). This control is used to determine any nonspecific effects that may be caused by the transfection reagent or process.

Untransfected cells control

Gene expression analysis should be carried out on cells that have not been treated to allow measurement of the normal, basal level of gene expression. Results from untreated cells can be used for comparison with results from all other samples. Untreated cells should be analyzed in every RNAi experiment.

Additional siRNAs for phenotype confirmation

A phenotypic effect caused by knockdown of a gene must be confirmed using at least one additional siRNA targeted against a different area of the mRNA.

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Guidelines for transfection of miRNA

microRNAs (miRNAs) are a class of endogenous small RNA molecules with similar characteristics to siRNAs. In recent years, it has been discovered that miRNAs play a role in many diverse biological processes such as development, differentiation, and apoptosis. Misregulation of miRNA expression is reported to be associated with several cancers and other diseases.

The miRNA system is an endogenous mechanism of regulation of gene expression. Mature miRNAs contribute to the regulation of endogenous genes, primarily by translational repression. In addition, miRNAs can mediate mRNA destruction by rapid deadenylation and/or decapping. Naturally occurring miRNA-binding sites are typically found in the 3' untranslated regions (UTRs) of target mRNAs. Their partial complementarity has made positive identification of true binding sites difficult and imprecise.

Transfection of miRNA mimics or inhibitors is a technique used to identify the targets and roles of particular miRNAs. miRNA mimics are chemically synthesized miRNAs which mimic naturally occurring miRNAs after transfection into the cell. miRNA inhibitors are single-stranded, modified RNAs which, after transfection, specifically inhibit miRNA function. Reduced gene expression after transfection of an miRNA mimic or increased expression after transfection of an miRNA inhibitor provides evidence that the miRNA under study is involved in regulation of that gene. Alternatively, the role of miRNAs in various pathways can be studied by examination of a specific phenotype following miRNA mimic or inhibitor transfection.

miRNA mimic or inhibitor transfection

When using 24-well plates, we recommend that cells are seeded in wells first, followed by addition of mimic/inhibitor–reagent complexes in order to ensure optimal mixing of cells and complexes. However, reverse transfection, where complexes are added to wells first and then cells are added on top of complexes, can be performed if desired. To perform a reverse transfection, simply change the order in which cells and complexes are added to the plate. 

However, for 96-well plates, we recommend using reverse transfection, since reverse transfection is rapid and convenient, and is frequently used for high-throughput formats. Reverse transfection is also optimal for cotransfection of miRNA mimic and miRNA inhibitor in 24-well plates. 

Plasmid DNA and miRNA mimic/inhibitor cotransfection

Many miRNA experiments involve cotransfection of an miRNA mimic and/or inhibitor together with a plasmid DNA vector in which miRNA-binding sites are fused to a reporter gene, such as luciferase.

Optimizing miRNA transfection

The amount of miRNA mimic/inhibitor needed to efficiently downregulate a target gene or inhibit miRNA function can vary greatly, depending on the miRNA, the cell line, and the chosen analysis method. To determine the concentration that provides optimal results, optimization experiments using varying mimic/inhibitor concentrations should be performed.

miRNA mimics can inhibit target protein expression at a final concentration as low as 0.5 nM. However, a higher concentration may be required, especially if performing downstream analysis at the protein level. miRNA inhibitors have been shown to inhibit miRNA function at a concentration of 50 nM. Lower inhibitor concentrations may also be effective.

In addition to optimization of concentration, time-course experiments may also be necessary to determine the optimal time after transfection for analysis of results. miRNA mimic or inhibitor effects often do not lead to an immediate change in transcript or protein levels.

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Performing appropriate miRNA control experiments

The use of appropriate controls is essential for the correct interpretation of results from experiments using miRNA mimics or inhibitors. Every experiment should include a suitable positive and negative control. Additional controls may also be necessary to enable interpretation of results or troubleshooting.

miRNA mimic experiments — positive control

Transfection of a positive control mimic can be used to confirm that the experimental system is working as expected (i.e., that the mimic is efficiently transfected and causes downregulation of the target). This control can also be used in optimization experiments where varying concentrations are used for transfection to determine the concentration that provides optimal results. A positive control should be routinely transfected in every experiment using miRNA mimics to confirm that conditions remain optimal.

miRNA mimic experiments — negative control

A negative control should be transfected in every experiment and will indicate if results are nonspecific. Comparison of results from the negative control with results from the miRNA mimic under study can be used to confirm that the observed results are specific to the miRNA mimic under study. Results from the negative control should also be compared to results from untransfected cells. Gene expression and phenotype should be similar in both untransfected cells and cells transfected with the negative control. Since miRNA mimics and siRNAs are chemically very similar and usually differ only in sequence, a negative control siRNA can also be used as a negative control miRNA mimic.

miRNA inhibitor experiments — positive control

In experiments involving transfection of miRNA inhibitors, detection of the inhibitor effect is often complicated by the presence of other miRNAs in the cell which interact with the same target gene. Cotransfection of mimic and inhibitor should result in an increase in expression when compared to the mimic alone. This confirms that the inhibitor is effectively inhibiting the mimic, resulting in upregulation of the gene target.

In experiments in which a reporter vector will be used for downstream analysis, the vector, mimic, and inhibitor should all be cotransfected together and, in parallel, the vector and mimic should be cotransfected (see “miRNA mimic experiments — positive control”). Transfection of vector, mimic, and inhibitor should result in an increase in expression when compared to transfection of vector and mimic. This confirms that the inhibitor is effectively inhibiting the mimic, resulting in upregulation of the reporter.

miRNA inhibitor experiments — negative control

A negative control should be transfected in every inhibitor experiment and will indicate if results are nonspecific. Results achieved after transfection of this control should be similar to results from untransfected cells. Comparison of results from the negative control with results from the inhibitor under study can be used to confirm that the observed results are specific to the inhibitor under study.

In experiments in which a reporter vector will be used for downstream analysis, the negative control should be a cotransfection of the inhibitor negative control and the reporter vector.

Transfection control

For optimal results, transfection efficiency should be as high as possible. When performing start-up experiments or working with a new cell line, it is necessary to perform multiple transfections under different conditions to determine the optimal conditions for maximum transfection efficiency. Transfection conditions that result in the greatest degree of cell death in comparison to transfection with a negative control can be maintained in future experiments. This control should be performed for optimization and start-up experiments and can be used as a routine transfection control in every experiment.

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References

1. Freshney, R.I. (1993) Culture of animal cells, a manual of basic technique. 3rd edition. New York: Wiley-Liss.
2. Ausubel, F.M. et al. eds. (1991) Current protocols in molecular biology. New York: John Wiley & Sons.
3. Sambrook, J., Fritsch, E.F., and Maniatis, T., eds. (1989) Molecular cloning — a laboratory manual. 2nd edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
4. Spector, D., Goldman, R.R., and Leinwand, L.A., eds. (1998) Cells: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
5. Jackson, A.L. et al. (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635.
6. Saxena, S., Jonsson, Z.O., and Dutta, A. (2003) Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells. J. Biol. Chem. 278, 44312.
7. Scacheri, P.C. et al. (2004) Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc. Natl. Acad. Sci. USA 101, 1892.
8. Sledz, C.A., Holko, M., de Veer, M.J., Silverman, R.H., and Williams, B.R. (2003) Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol. 5, 834.
9. Semizarov, D., Frost, L., Sarthy, A., Kroeger, P., Halbert, D.N., and Fesik, S.W. (2003) Specificity of short interfering RNA determined through gene expression signatures. Proc. Natl. Acad. Sci. USA 100, 6347.
10. Persengiev, S.P., Zhu, X., and Green, M.R. (2004) Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 10, 12.

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