DNA, RNA, and Protein as Transfection Substrates

* This product is for research use only. Not intended for use in the treatment or diagnosis of disease.

To meet the research requirements, DNA, RNA, and protein can be explored to transfect diverse cells, with their advantages and limitations. According to different cell types, experimental backgrounds, or technical applications, the transfection substrate can be chosen rationally, and then the specific transfection conditions can be optimized.

Plasmid and DNA Transfection

DNA is the most commonly used transfection substrate in the laboratory because it is stable, easy to construct and prepare, and cost-effective to produce. During transfection, high-purity DNA should be used as far as possible to avoid all impurities including lipids, proteins, salts, phenols, etc., and the endotoxin should be removed. The DNA purity can be determined by measuring OD 260/280 value, which should be between 1.7 and 1.9. High or low values indicate impurities and should not be used in transfection experiments.

Linearization or superhelix can affect transfection results: superhelix plasmids are much more efficient than linear DNA, especially transient transfection, however, the integration rate of linearized DNA transfection was higher. If the plasmid is too large, transfection will be more difficult. After all, relatively dense, small foreign bodies are more likely to be endocytosed by cells. Some transfection reagents can provide some components that promote DNA aggregation, making the formation of DNA transfection complexes denser and easier to transfect. The quality of purified plasmids will also affect the transfection efficiency, so high-quality plasmids must be selected. Of note, although too little DNA will lead to low transfection efficiency, too much DNA will also reduce the transfection efficiency.

Promoter selection is very important for the effective expression of transfected genes. Although it has little influence on the transfection process itself, it has a subtle influence on the transfection result.

RNA Transfection

RNA transfection provides another means of research exploration different from DNA transfection - direct transfection of mRNA, viral RNA, RNA oligonucleotides, siRNA and even ribosomes into cells. RNA transfection results in faster and more direct results than DNA transfection because direct translation requires neither transcription nor nuclear uptake - the protein is expressed directly in the cytoplasm. The structure of RNA itself has an impact on transfection. Mammalian mRNA molecules have a 5'-end cap and a 3'-end Poly A tail that both improve mRNA stability and help the ribosome bind to the mRNA for translation. The RNA transfection process is complicated by many adverse influences, such as contamination by RNase and degradation of RNA after entering the cell. The degradation of RNA after transfer into the cell is more important in the RNA transfection process. If the transfected RNA is heavily degraded after entering the cell body, the experimental results will be very inaccurate.

RNA transfection has many advantages

  • It expresses proteins in a completely promoter-independent manner, without the risk of genomic integration.
  • It is well suited for transfection of slow-growing or non-dividing cells.
  • The protein expression during transient transfection lasts for a limited period, avoiding the accumulation of toxicity.

Protein Transfection

Proteins are a class of highly versatile biomolecules that play a key role in determining cell function. In therapy, proteins provide a powerful and versatile approach to disease treatment because they can be delivered into cells to replace dysregulated proteins or manipulate cellular signaling processes. Proteins can also serve as highly specific recognition and signaling components for probing cellular molecules to advance fundamental understanding of biology or to discover new paradigms for disease diagnosis. The benefit of using proteins for transfection is that they often have an immediate impact on the cell and allow better control of numbers. This challenge arises from the cellular impermeability of most proteins and their propensity to degrade in the complex biological microenvironment. Due to the structural diversity of proteins (i.e., their degree of charge, size, and hydrophobicity), each protein has its unique challenges in transfection into cells and in maintaining activity.

Multiple strategies have been developed to improve protein stability and cellular uptake. These strategies include protein surface engineering to express large amounts of positive charge, chemical modification with polymers or cell-penetrating peptides/proteins, and optimization of protein transfection with preparations containing lipids, liposomes, or nanoparticles. However, the transfection conditions required for protein transfection are expensive and more complex than for DNA and RNA.

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