DNA transfection is a main tool for current genetics as well as cell and molecular biology studies. Understanding the underlying mechanisms and the different parameters affecting transfection is crucial for optimal, reproducible, and trustable results. Following these seven tips will ensure reliable results.

• Adapt the transfection conditions to your experiment. Transfection conditions should be optimized according to the cells, the size of the plasmid, the number of plasmids, the expression pattern of the gene of interest, the culture vessel, and the transfection reagent. Reoptimize the conditions for each new cell line.

• Transfect healthy cells. Passage cells at least twice after thawing to allow recovery before transfection, and use cells at low passage number (< 20 passages). Discard overconfluent cells. Regularly check for mycoplasma contaminations. Seed cells the day before transfection accordingly to the confluency recommended by the transfection reagent provider.

• Follow the transfection reagent protocol. Some reagents are inhibited by serum or antibiotics, while others may be used in serum- and antibiotic-containing medium, hence reducing the risks of toxicity and the number of steps in the protocol. Check the recommended cell confluency, as well as the recommended DNA amount and reagent volume.

• Work with high-quality DNA preparation. Check for RNA contamination by agarose gel electrophoresis and ethidium bromide staining. Measure UV absorbance at 280 nm. OD260/280 ratio should reach at least 1.8. Resuspend the plasmid in deionized water or TE buffer at a concentration of ca. 1 µg/µl. Aliquot the plasmid preparation and store at -20°C to avoid freeze/thaw cycles.

• Check serum quality. Some serum lots may inhibit drastically transfection efficiency, hence resulting in lower silencing efficiency. Check transfection efficiency of different serum lots before purchasing a new batch of serum. Also ensure that the medium used allows for efficient transfection, as some media compound may decrease transfection efficiency.

• Minimize cytotoxicity by using low DNA amount and low transfection reagent volume. Check that the target gene does not affect cell viability. Analyze transfection at an earlier time point (24 h after transfection instead of 48 h for instance).

• Use appropriate controls. Use a reporter gene to set up and optimize transfection conditions, as those may vary depending on the cells to transfect. Various reporter systems are commercially available: Renilla Luciferase and GFP (Green Fluorescent Protein) are the most commonly used.

Polyplus-transfection created a database where scientists can find optimal conditions to transfect their cells:

Search in the Polyplus-transfection database