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6 Tips for Optimal Transfection

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Transfection is an essential cell biology technique by which one can introduce nucleic acids (DNA & RNA) into eukaryotic cells. It is particularly useful when one wants to express genetically engineered protein in cells (usually via plasmid DNA transfection) or deplete endogenous protein (via siRNA transfection).

Broadly speaking, three transfection methods are popularly used: chemical transfection, electroporation (or nucleofection), and lenti/retroviral transfection. The first two methods are considered as “transient transfection”, because the amount of the introduced nucleic acids in cells decreases over time due to degradation and dilution. On the other hand, viral transfections incorporate DNA into an endogenous chromosome, which replicates over the cell cycle, and therefore is considered as “stable transfection”. In this guide, I will focus only on transient transfection methods as in my experience those are trickier than viral transfection.

There are many commercial kits for chemical transfection and electroporation. Whether you follow a protocol from the manufacturer or from a paper, you will often find it difficult to get a satisfying result without optimizing the protocol to your needs. The following is a list of what I think are the most useful tips for successful transfection, learned through years of experience and troubleshooting transfection experiments.

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Chemical transfection vs electroporation

There are a number of advantages of chemical transfection. First of all, it doesn’t require a device and cuvettes. Considering that a very basic electroporation device is around $10,000, this can be a huge advantage. Secondly, there is greater flexibility in the number of cells required for transfection. With electroporation, the number of cells used is largely dependent on the size of cuvette.

In which case why would one want to use electroporation? Firstly, electroporation works for certain cell types that are hard to transfect with chemical transfections (e.g. some primary cell lines). Second, in my opinion, the protocol is simpler, as you can perform electroporation when splitting cell culture.

Some additional tips: 
  • Most commercial chemical transfection kits are designed for either DNA transfection or siRNA transfection, but I’ve seen many people saying that any kit can be used for both applications. 
  • Most companies offer free samples that you can use to try out. 
  • Electroporation devices can also be used to deliver other membrane-impermeable molecules into cells (e.g. Alexa dyes).

Do optimization

First, try the manufacturer’s protocol. Some manufacturers also provide cell-type-specific protocols, so check their websites. If you end up seeing devastating cell death (which often happens) or very low transfection efficiency, then you should optimize. If you are doing chemical transfection, vary DNA amount, DNA-to-reagent ratio, transfection-to-assay time, and the presence of serum & antibiotics in your growth media (unless the protocol says it really doesn’t matter). The most important factor, in my opinion, is the amount of DNA and reagent. I always got better results when I used less DNA and reagent than the amount suggested in the manufacturer’s protocol. If you are using electroporation, you could try different amounts of DNA, voltage, and duration of pulse.

Do validations

You can use RT-PCR, western blot, FACS or imaging to evaluate transfection efficiency. My favorite validation method is to transfect with GFP plasmid or fluorescent control siRNA and quantify the fraction of cells showing fluorescence signal by using a simple fluorescence microscope with 10x objective.

Use high quality DNA

Plasmids extracted from E.coli culture may be contaminated by endotoxin, which is detrimental to cell viability in transfection experiments. I highly recommend using midi- or maxi-scale DNA extraction kits (instead of miniprep) which involves more extensive washing steps and ethanol precipitation steps.

Use healthy cells. The viability and health of cells are also important factors influencing transfection efficiency and post-transfection viability of the cells. 
  • Limit passage number; I generally do not maintain a cell culture for more than 2 months. 
  • Give cells sufficient time to recover from stresses. After thawing a new vial of frozen stocks, I recommend passaging cells a couple times prior to use. I would also ensure that the cells have at least grown overnight following splitting prior to use for chemical transfection.
  • Be cautious of contamination, especially if you don’t use any antibiotics in your growth media, as it affects the transfection efficiency. Do mycoplasma test regularly (it’s very simple to perform), and if you get a positive on the test, or see small objects (a few micrometers) floating around under the microscope, don’t hesitate to throw away your culture and thaw a fresh vial of frozen stocks, and replace your media, PBS, and trypsin. 
  • Don’t let your cell culture become over-confluent. Passage the cells before they reach ~90% confluency.

Do control experiments

This applies to any biology experiment, but is particularly important for siRNA transfection experiments. Do transfections with control siRNA (siRNA that doesn’t deplete any protein) to assess the toxicity or any side effect of the transfection.

I hope you find these tips useful. If you are still not satisfied with your transfection results, consider using viral transfections, replacing the promoter in your plasmid with an inducible or weaker promoter (to reduce the toxicity due to over-expression), or switching to other cell types if possible.