An Overview of Transfection: methods, tools, and protocol.

What is transfection?

Transfection is a powerful analytical tool enabling study of gene function and regulation and protein function in cells. The most commonly used method can be broadly classified into three groups, biological, chemical, and physical. Each method has its own advantages and disadvantages, and is suitable for different types of cells and experiment conditions.

Physical Methods

  • Microinjection

Directly injects nucleic acid into the cytoplasm or nucleus. It is a very simple and straightforward method, but it needs expensive instruments and usually max of only 100-200 cells in single treatment.  

  • Biolistic transfection

This method was usually used in plant cell transfection, since it can penetrate across cell walls. And the greatest advantages of this method is its ability to overcome physical barriers like epidermis, and can transfect large quantities of cells per single use. However, the weakness of it may lies in the cost.

  • Electroporation

High-voltage electric shocks may induce temporary disruption in cell membrane permeability which allows translocation of different molecules into the cell. And by adjusting the pulse amplitude, electroporation can be modified to affect only membranes of organelles.  

  • Optical transfection

Optical transfection has comparable efficiency for its ability to transfect single cells and it is simpler than other methods with similar capability. This method was used a lot in studies comparing effects of mRNA injection to the dendrite and neuronal cell bodies.

Biology Methods

  • Adenoviruses

Adenoviruses are viruses that have double-stranded DNA structure and are used for gene delivery. Since the infection adenoviral DNA is not integrated to the host cell chromosomes, it is not possible to induce prolonged protein expression. By using this method, the expression level of introduced protein usually are very high at the beginning but quickly weaken after several weeks.    

  • Adenoviruses-associated Virus

This type of virus is a small, single-stranded DNA, non-enveloped and replication-defective. The advantage of this method is that the used virus is shown to be weakly immunogenic when compared to adenoviruses.

  • Retroviruses

Retroviruses, especially Lentiviruses (HIV), are widely used due to their ability to infect a variety range of cell types. It has been approved in the US in treatment with acute lymphoblastic leukemia.

Chemical Methods

  • Calcium Phosphates

Calcium phosphate is positively charged molecule that can form complex with negatively charged DNA in order to introduce DNA into mammalian cells. Different calcium phosphate transfection can trigger either endocytosis or phagocytosis. Even though this method is comparably inexpensive, the disadvantage of this method is that it mutates DNA at a high frequency and it can only tolerate very small pH changes.

  • Cationic Lipids

This technique uses a positively charged lipid/liposomes to interact electrostatistically with negatively charged phosphate residues of DNA and cell membrane. The underneath mechanism is endocytosis first, and then followed by complex release into the cytoplasm. It can be used to various cell lines and it’s very efficient. It can also deliver a broad range of molecular mass to the cell, such as DNA, RNA, and protein, and is suitable for either stable and transient transfection.

  • Magnetic Beads

This method uses magnetic force/field in order to introduce DNA is called magnetofection. This is a rapid and non-viral method used only for adherent cells, but it adapt for many types of nucleic acids. Its advantage is that it compromise low amount of DNA. This method cause very minimum membrane damage.

  • Cationic Polymers

Many types of polymers were successfully tested for gene delivery and reproductive medicine. It is more efficient DNA condensation in comparison to cationic lipids. They could be good carriers for plasmid DNA, siRNA, and mRNA, and is suitable for many different cell types. But cytotoxicity of carriers is a big concern. A good carrier should be non-toxic for treated cells and has high transfection efficiency. There are already some modifications of polymer structure such as pegylation allows to decrease the polymer cytotoxicity through introduction of poly(ethylene) glycol into carriers structure.


Recommended Product

RM09014 HighGene Transfection reagent


Fus-Kujawa, A., et al., An Overview of Methods and Tools for Transfection of Eukaryotic Cells in vitro. Front Bioeng Biotechnol, 2021. 9: p. 701031.


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