Transfection
Cationic or ionisable lipids condense DNA or RNA into lipoplexes that are endocytosed; the workhorse method for adherent cell lines, with efficiencies of 50–90% in HEK293 and HeLa and a reagent-to-nucleic-acid ratio that has to be titrated for each line.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Lipofection
Cationic or ionisable lipids condense DNA or RNA into lipoplexes that are endocytosed; the workhorse method for adherent cell lines, with efficiencies of 50–90% in HEK293 and HeLa and a reagent-to-nucleic-acid ratio that has to be titrated for each line.
How it differs from the standard method
- Form complexes in serum-free reduced-serum medium; serum blocks particle formation but is fine on the cells afterwards.
- Titrate reagent volume per µg of DNA (commonly 1.5–4 µL/µg) against both efficiency and viability.
- Transfect in antibiotic-free medium: transiently permeable cells take up antibiotics they normally exclude.
- Replacing the medium 4–6 h after addition recovers viability at no cost in efficiency.
- Different formulations are sold for DNA, siRNA and mRNA; they are not interchangeable.
1Getting a nucleic acid across two membranes
DNA and RNA are large polyanions. A cell membrane is a hydrophobic sheet with a net negative surface, so nothing about a plasmid lets it drift in.
The plasmid does not replicate
Expression peaks around 24–48 h and then dilutes away as the cells divide, which is the definition of a transient transfection.
Efficiency is a fraction of cells
Report the percentage of live cells that express, measured against an untransfected control, not a visual impression.
Every method kills cells
Cationic reagents and electric pulses are both toxic; a fair experiment reports viability beside efficiency.
2What the read-out physically is
A fluorescent reporter read by flow cytometry gives two numbers that matter: the fraction of live cells above the autofluorescence of an untransfected control, and how bright those cells are. The fraction is about how many cells took up and expressed anything; the brightness is about how many copies each one got and how strong the promoter is.
3How it goes wrong
- Complexes made up in serum-containing medium: serum proteins coat the lipid and the DNA before a particle can form.
- Too much reagent: efficiency plateaus, toxicity does not, and the surviving cells are a selected subpopulation.
- Transfecting a confluent sheet, or cells that were split into antibiotic-containing medium that morning.
- An endotoxin-rich or salt-rich plasmid prep: endotoxin is toxic to the cells, salt makes an electroporation arc.
- Reading the plate at whatever time is convenient, so the transient peak is missed in one direction or the other.
- No untransfected control, so the gate sits in the negative population and a few per cent of autofluorescence is read as expression.
- Treating per cent positive as the whole story when the experiment depends on how much protein each cell makes.
4Choosing a method
- Cationic lipid
- Highest efficiency in common adherent lines, works with DNA, mRNA and siRNA, expensive per well, and the reagent is the main toxicity.
- PEI
- Pennies per litre and the standard for large-scale transient protein production in HEK293 and CHO suspension cultures; needs careful N/P ratio and cell-density optimisation.
- Calcium phosphate
- Cheapest of all and historically important; very sensitive to pH, mixing order and the age of the precipitate, so reproducibility is poor.
- Electroporation / nucleofection
- Works where chemistry fails (suspension lines, primary T cells, stem cells) and is the standard route for ribonucleoprotein editing reagents; needs special solutions, a device and more cells per sample.
Key terms
Practise these as flashcards →The particle formed between nucleic acid and a cationic lipid or polymer; its size and charge decide uptake.
Moles of polymer amine nitrogen per mole of DNA phosphate: the quantity that is actually optimised when a PEI transfection is titrated.
The step at which an internalised complex breaks out of the endosome; usually the limiting step for chemical transfection.
Expression from a plasmid that does not replicate or integrate, so it fades over days as the cells divide.
Electroporation with cell-type-specific solutions and pulse programmes designed to deliver cargo to the nucleus with high survival.
Adding complexes to the plate and then seeding cells into them, rather than onto an attached monolayer.
The fraction of cells receiving both of two plasmids added together; it is high per transfected cell, because uptake is all-or-nothing per cell.
The fluorescence threshold taken from an untransfected sample, above which a cell counts as positive.
When to use it
- To express a protein or a reporter for a few days: a luciferase assay, a localisation experiment, a pull-down from over-expressed bait.
- To deliver siRNA or mRNA, where the cargo acts in the cytoplasm and transient delivery is all that is needed.
- To produce virus: packaging a lentiviral or AAV vector is itself a multi-plasmid transfection of HEK293T cells.
- To deliver Cas9 as a ribonucleoprotein or as plasmid DNA when a short, self-limiting burst of nuclease is wanted.
- To make large amounts of secreted protein transiently in suspension HEK293 or CHO cells, where PEI is the standard reagent.
Compared with alternatives
Prefer a viral vector for primary cells, non-dividing cells and anything needing stable expression or a single integrated copy; transfection wins on speed, cost and biosafety paperwork.
If the experiment runs for weeks, or needs every cell to express the construct, select a stable line instead of repeating a transient transfection.
Knockdown by siRNA is a transfection whose cargo is a duplex rather than a plasmid; the delivery problem is the same, the dose-response and controls are not.
Most promoter and pathway reporter assays are read a day or two after a transient transfection, with a co-transfected normalisation reporter to correct for how well each well took up DNA.
Flow is how transfection efficiency is actually quantified: per cent positive of live cells, with the gate set on an untransfected control.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Cationic or ionisable lipids condense DNA or RNA into lipoplexes that are endocytosed; the workhorse method for adherent cell lines, with efficiencies of 50–90% in HEK293 and HeLa and a reagent-to-nucleic-acid ratio that has to be titrated for each line.
- Form complexes in serum-free reduced-serum medium; serum blocks particle formation but is fine on the cells afterwards.
- Titrate reagent volume per µg of DNA (commonly 1.5–4 µL/µg) against both efficiency and viability.
- Transfect in antibiotic-free medium: transiently permeable cells take up antibiotics they normally exclude.
- Replacing the medium 4–6 h after addition recovers viability at no cost in efficiency.
- Different formulations are sold for DNA, siRNA and mRNA; they are not interchangeable.