Low Transfection Efficiency? 5 Points You May Not Have Considered
Updated: Aug 28
All researchers working with cell transfection methods, whether in academia or in industry, know about the pains involved in optimising cell transfection. Sometimes some cells just seem to be more resistant to transfection than others, often for frustratingly vague reasons.
So we've put together a 5 point list covering the major factors impacting transfection efficiency. Use these points as a guide to help you start to optimise your experiments and get more transfected cells.
Is poor transfection efficiency wasting time in your high throughput processes? We can help. Get in touch.
The 5 major factors that affect transfection efficiency are
1. Cell viability
2. Cell type
3. Transfection Methods used
4. Confluency and replication state
5. Nature of insert or expressed protein
1. Cell Viability
When considering the transfection process as a whole, transfection efficiency and cell viability are intrinsically linked - there's little point in having high transfection efficiency no cells survive.
Prior to transfection, cells should be >90% viable and been given time to recover from passaging.
Factors impacting viability are manifold; if you're seeing low viability of cells prior to transfection, check the following:
Nutrition: that the media, serum and supplementation requirements are the right ones for your cell line. Each cell line has its own unique requirements.
Contamination: Contamination can be biological or chemical. Biological contamination of cell lines by bacteria, viruses, mould, yeast, and mycoplasma can occur and impact negatively on the health of your cell. With bacteria, a sudden decrease in pH is often seen; with yeast and mould the pH rises in the later stages of infection. Other warning signs include a turbid cell culture or pronounced cell death. Biological contamination can be prevented by good aseptic technique.
Cross-contamination by other mammalian cell cultures is a widespread and under-diagnosed issue - a study in 1976 tested 246 cell lines and found that a stunning 30% of these were incorrectly labelled. If you're uncertain, look at isotyping, DNA fingerprinting and karyotyping of cells to diagnose the problem.
Cells can also be contaminated by chemical factors (detergents, endotoxins, foreign matter in media).
If you are planning on using antibiotics to treat bacterial contamination, first check that these substances are not toxic at therapeutic doses to your particular cell line.
Toxicity: Potential toxicity to the cell by your transfectant should be considered as it impacts profoundly on cell viability. If you're trying to express a potentially toxic protein, your system should be inducible and non-leaky.
If you’re having issues with cell death in expressing a toxic protein, consider switching to a different promoter-inducer system.
The viability of cells is something our Inish Analyser technology can routinely help determine, to isolate only the best of your transfected cells.
2. Cell type
Choice of cell type will ultimately depend on the function of the experiment. For recombinant protein production the choice of cell line may be all but unimportant; for gene expression characterisation, it could be critical. When choosing, bear these points in mind.
Established cell lines are generally regarded as easier to work with, but their transformation into immortalised cells could represent genetic changes which make them unrepresentative of normal populations, which restricts their use for certain topics.
In our work on improving gene transfection efficiencies for high throughput experiments, we often speak with representatives in industry and academia about their problems with transfection; from this, we know that types generally regarded as 'difficult' to transfect include primary cells, stem cells, and cells in suspension.
3. Transfection Methods Used
Choice of transfection method is also crucial to the efficiency of the process. Transfection methods can be physical or chemical, and nucleic acid uptake can also be induced by viral transduction of cells.
There are dozens of choices of each type of product available on the market currently. The two most popular are viral transduction and electroporation, but lipid-based transfectants are also popular. Traditionally, there was somewhat of a trade-off between transfection efficiency and cell death - particularly with methods such as electroporation - and to a certain extent, it still exists.
Choosing a transfection method isn't exactly straightforward, as the ideal choice depends on what outcome you want (i.e. stable vs. transient transfection) or on what you're working with. Your target location- the nucleus or the cytoplasm - can influence your choice, as can a need for co-transfection or purification requirements of the resulting cells. There are also exceptions to rules; in most cases, serum enhances transfection efficiency; unless you're working with lipofectamine, in which case it interferes with the process.
With all these factors it's no wonder choosing a transfection process is so difficult, and quite often researchers will simply go with the lab default rather than determining what is really optimal for their needs.
How to choose transfection methods is something we'll touch again on later in the series, but is something which needs to be carefully considered before undertaking transfection of cells.
4. Confluency and replication state
Cell confluency and replication stage are factors intrinsically linked to each other that affect the efficiency of your transfection protocols.
This is because cells that are actively dividing take up DNA more readily than stationary phase cells. Controlling cell confluency is how cells can be kept in an actively dividing state. If cells are allowed to become fully confluent, they begin to exhibit contact inhibition and cease actively dividing - this limits their ability to uptake nucleic acids and negatively impacts transfection efficiency.
Generally, it is recommended that passaging of cells should take place once or twice a week. The optimal level of confluency before transfection varies depending on cell type and should always be determined ab initio if data cannot be found; very generally, it lies around 70-80%.
5. Nature of Insert
The nature of what you are inserting into the cell effects the transfection efficiency in several ways. The size and charges of your transfectant in combination with your method of choice can impact the efficiency of transfection. For example, lipid-based transfectants readily transect negatively charged molecules, but uncharged molecules are a challenge.
Trying to improve your transfection efficiency? So are we. Our Inish Analyser can use impedance spectroscopy for label-free multiparametric analysis and sorting of cells - and the monitoring of membrane conductivity allows for unparalleled separation of your ideal transfected cell pool. We're currently looking for research partners working with mammalian or plant cells, so if this interests you get in touch with us for a chat.
Have you worked with cells which you found particularly difficult to transfect?
Know of any other factors influencing transfection efficiency?
We welcome your comments below, or you can get in touch.