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Predicting Transfection Efficiency – Why is it So Important?

Ensuring efficient transfection is essential for gene therapy, CRISPR/Cas9, and many other applications in industry and research laboratories. However, many factors affect transfection efficiency. Furthermore, the methods available to assess this efficiency can be laborious and time-consuming. If you're involved in gene expression and modulation research, you may be familiar with these issues. But as always, science evolves, and new alternatives arise to facilitate our work and accelerate our discoveries.

What is transfection?

For those just starting transfection experiments, let's clarify some basic concepts before delving into the topic.

Transfection is a method that allows inserting foreign nucleic acids (DNA and RNA) into eukaryotic cells, [1]. With this powerful technique, researchers can modify the host cell's genetic makeup. The applications are endless; you can study disease's mechanisms, discover biomarkers for diagnosis and prognosis, or develop gene therapies to treat genetic diseases [1].

Examples of applications of transfection techniques [1]:

  • Gene silencing

  • Stable cell line generation

  • Virus production

  • Large-scale protein production

  • Stem cell reprogramming and differentiation

Types of transfection

There are two types of transfection:

  • Stable – Stable transfection involves integrating the foreign nucleic acid into the host cell nucleus maintaining its expression in the long term, [1].

  • Transient – This type of transfection does not involve integrating nucleic acids into the host cell genome. For instance, you can use a plasmid or oligonucleotides to transfect nucleic acids. However, gene expression will be lost with cell replication, [1].

Briefly, stable transfection is helpful if you need large amounts of protein production. In contrast, transient transfection is more useful to investigate the short-term effects of a gene knock-out or knock-in, [1].

The options for delivering the genetic material into the host cells include vectors like viruses or plasmids, [1]. Common transfection techniques are calcium phosphate precipitation, lipofection, and electroporation, which increase cell permeability through an electrical field, [1].

Why is transfection efficiency important?

If you're a researcher working with cell transfection, you know how challenging it can be to optimize the process. Efficient transfection is key to the experiment's success. However, some cells seem to be more resistant than others, and sometimes you can't identify the reason.

With the great variety of approaches and delivery methods, transfection efficiency can vary [1]. Other factors like cell type and viability, confluency, number of passages, amount of nucleic acid, and medium quality can also influence transfection efficiency [2].

So, choosing a proper method to assess this efficiency is a critical step for your transfection experiment [1].

Problems with quickly identifying successfully transfected cells

Following transfection using the chosen method, assessing transfection success usually involves:

  • Transfering the cells to a 96-well plate for culture.

  • Incubating the cells for 24 to 48 hours. After that, cells start to express the gene of interest, such as a specific cell receptor on the surface of the membrane.

  • Adding fluorescent dyes or magnetic beads to sort the successfully transfected cells.

This method can be time-consuming since you have to wait for 24-48hrs before seeing if your experiment worked. Also, preparing the samples for cell staining can be costly and laborious.

But what are the alternatives for ensuring transfection efficiency and optimising your process?

Cellix’s Transfection Efficiency Prediction Assay

After electroporation, you can have four cell populations:

  • Alive but closed

  • Dead

  • Damaged

  • Alive and open, the useful set

In traditional Cell Analysis assays, these cell populations are lumped together (excluding the dead ones). Why? Well, after electroporation, the cell membrane has been opened. With traditional Cell Analysis assays, viability stains would indicate that these cells are dead – but they’re not! In fact, these are the cells that you are most interested in....the ones that have had their cell membranes’ opened to successfully receive the gene insert.

With the Inish Analyser, you can predict transfection efficiencies minutes after transfection - before you go to the hassle of plating your cells; no need to wait 24-48 hours to find out! This saves you time and improves your workflow. And, as no labeling is required, it also reduces the assay's cost.

1. Quantifying the number of cells that have successfully had their membranes open and are most likely to be successfully transfected. Results are obtained in minutes and presented as a Transfection Efficiency Prediction.

2. Observing the different cell populations after transfection.

  • Cells that are alive but closed indicate transfection failure since the membrane failed to open to receive the vector/DNA.​

  • Cells that are alive and open are your proof of a successful transfection since the cell membrane has been successfully opened. These cells can uptake the vector.

3. Observing the recovery of the cell membrane after electroporation, cell squeezing (French press), microinjection, and other physical methods.

The principal benefits of this technique are:

  • Prediction of your transfection efficiency before you go to the hassle of plating your cells, saving you time and improving your workflow.

  • No labeling is required, reducing the assay's cost.

Is poor transfection efficiency wasting your time? We can help. Contact Cellix to book an online demo or request a quote.


1. Chong ZX, Yeap SK, Ho WY. Transfection types, methods and strategies: a technical review. PeerJ [Internet]. 2021 Apr 21;9:e11165–e11165. Available from:

2. Fisher T. Factors Influencing Transfection Efficiency [Internet]. [cited 2021 Jun 29]. Available from:


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