You can use DNA, RNA or proteins to transfect your cells, and each has its own advantages and disadvantages. Additionally, certain cell types or applications may only work with a specific transfection substrate, and you will often need to reoptimize your transfection conditions when switching substrates. More details on each transfection substrate are provided below.
Plasmid and Linear DNA Transfection
Plasmid DNA is commonly used for transfection (although linear DNA can also be used). In both cases, you should ensure that your DNA is as pure as possible, with any contaminating lipids, salts, proteins, nucleotides or other factors removed via DNA purification. The best way to obtain high quality DNA is by using a purification method based on anion exchange technology which includes an additional endotoxin removal step. This helps to ensure optimum transfection efficiency and cell viability. When transfecting DNA purified with silica membranes, there is a high risk, on poor transfection efficiency and cell viability, further downstream results might not be trustable.
DNA is the substrate of choice for a wide range of applications, as it is relatively stable, easy to handle and cost effective to produce. DNA vectors are also the best option if you are looking to achieve stable transfection via incorporating your construct into the target cell’s genome. However, as DNA constructs must make their way into the nucleus to induce gene expression, they are slower to drive protein production than mRNA (which exerts its effects as soon as it enters the cytoplasm, see below). In all cases the toxicity of the expressed protein has an impact on the transfection result, no matter if DNA, mRNA or the protein itself was transfected. When transfecting plasmid DNA, also the plasmid backbone and the promoter can have an influence on the transfection result.
The RNA used for transfection can take several forms, depending on the application. For example, mRNA is an effective substrate for inducing rapid protein expression, as it enables results to be achieved more quickly than using DNA. It is also possible to transfect cells with regulatory or non-coding RNAs to modify the expression of endogenous genes (e.g. gene knockdown via RNA interference).
In general, RNA transfection has a higher efficiency than DNA transfection as it just needs to enter the cytoplasm. Furthermore, with DNA-sensitive cells, cell viability is often much better when using RNA. As already said before, the toxicity of the encoded protein has an impact on the transfection result as well. However, protein production may be lower overall, as mRNA is more unstable than DNA and no new RNA is produced by the cell following transfection. The general instability of RNA can also make it more challenging to work with. Meanwhile, production can also be more cumbersome (as the mRNA must be transcribed from a DNA template, introducing an extra step) and/or expensive (if the RNA will be chemically synthesized).
Some applications are not well-suited to RNA transfection. For example, to create induced pluripotent stem cells (iPSCs) using mRNA, you must deliver it repeatedly over time. In addition, RNA cannot be used for stable transfection, as it will not be integrated into the host cell’s genome.
Protein transfection is used for specific applications, such as the introduction of Cas9 enzymes for gene editing via CRISPR, and the use of transcription factors to reprogram cells to generate iPSCs. The advantage of using proteins is that they usually exert their influence on the cell immediately and the quantity can be controlled much better. However, proteins vary greatly in terms of size, shape and charge, so transfection conditions must often be specifically optimized on a per-protein basis. Another practical consideration is that proteins must either be made and purified or purchased from a supplier – both options can be costly, especially when compared to generating DNA or RNA substrates.