Guidelines for transfection

Guidelines for transfection of DNA

DNA quality strongly influences the results of transfection experiments. The best results are achieved when plasmid DNA of the highest purity is used for transfection.

Endotoxins, also known as lipopolysaccharides (LPS), are cell membrane components of Gram-negative bacteria (e.g., E. coli) that are released during the lysis step of plasmid preparation and are often copurified with plasmid DNA. The presence of endotoxins in plasmid DNA can result in sharply reduced transfection efficiencies, particularly in primary cells and sensitive cell lines. For transfection of endotoxin-sensitive cells (e.g., primary cells, suspension cells, and hematopoietic cells) or for highest transfection efficiencies and lowest cytotoxicity, we recommend using DNA purified using a commercially available kit specifically designed for endotoxin removal. Such kits help to ensure optimal transfection results.

The configuration of a DNA molecule used for transfection influences the efficiency of transfection. Transient transfection is most efficient with supercoiled plasmid DNA. In stable transfection, linear DNA yields optimal integration into a host cell’s genome although uptake into cells is lower compared with plasmid DNA.

If the gene product of DNA transfected into a cell is toxic to the cell, its expression will lead to cell mortality. In such cases, it may be necessary to use either a weak or an inducible promoter to limit the damage caused to cells due to expression of a toxic gene product.

Several methods for transfection of nucleic acids have been developed using DEAE-dextran, calcium phosphate, electroporation, liposomes, non-liposomal lipids, and activated dendrimers. The choice of transfection technology can strongly influence transfection efficiency. Ideally, transfection should be fast and easy to perform, give high efficiencies and reproducible results, and cause minimal cytotoxicity.
For the best results and the most efficient transfection, an optimal ratio of DNA to transfection reagent must be determined for each cell line–plasmid combination. Therefore, it is vital that optimization trials are carried out for each new cell line–plasmid combination used.
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