When cells are transiently transfected with plasmids, the DNA is introduced into the nucleus of the cell, but does not integrate into the chromosome. This means that many copies of the gene of interest are present, leading to high levels of expressed protein. Transcription of the transfected gene can be analyzed within 24–96 hours after introduction of the DNA depending on the construct used. Transient transfection is most efficient when supercoiled plasmid DNA is used. siRNAs miRNAs and mRNAs can be used for transient transfection and are effective in the cytoplasm, without the need to be transferred to the nucleus.
With stable or permanent transfection, the transfected DNA is either integrated into the chromosomal DNA or maintained as an episome. Stable integration of plasmid DNA into the genome is a rare event. Stably transfected cells can be selected by co-transfection of a second plasmid carrying an antibiotic-resistance gene or by providing a resistance gene on the same vector as the gene of interest. siRNA and miRNA can only be stably transfected when they are delivered as short hairpin transcripts made from a selectable DNA vector. However, RNA molecules per se cannot be used for stable transfection.
Although linear DNA results in lower DNA uptake by the cells relative to supercoiled DNA, it yields optimal integration of DNA into the host genome. Cells which have successfully integrated the DNA of interest or have maintained episomal plasmid DNA can be distinguished by using selectable markers. Frequently used selectable markers are the genes encoding aminoglycoside phosphotransferase (APH; neoR gene) or hygromycin B phosphotransferase (HPH). Other selectable markers are the genes encoding adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hymidine kinase (TK), or xanthine-guanine phosphoribosyl transferase (XGPRT; gpt gene).
Fast-forward and reverse transfection
In fast-forward transfection, plating and transfection of cells are performed on the same day. DNA or siRNA is diluted in culture medium without serum. Transfection reagent is added to the diluted nucleic acid (NA) to produce reagent–NA complexes.
The cells are seeded and then complexes are added directly to the freshly seeded cells. Whereas, in a traditional transfection, cells are plated 24 hours prior to transfection. Cells are seeded in culture medium containing serum and antibiotics the day before transfection and incubated under normal growth conditions. The next day, DNA or siRNA is diluted in culture medium without serum. Transfection reagent is added directly to the diluted DNA or siRNA to produce reagent–NA complexes. During complex formation, the medium on the cells is changed, before the complexes are added to the cells (see figure Fast forward and reverse transfection).
Fast forward and reverse transfection
In standard or fast-forward transfection, cells are added to plate wells first, followed by transfection complexes. In reverse-transfection, nucleic acid is added to plate wells, followed by transfection reagent. Cells are added after complex formation in the wells, hence the term “reverse transfection” (see figure Steps in fast-forward and reverse transfection).