Tips for transfecting cells

Whether you are transfecting cells to over-express a gene, to knock down a gene, or to measure some cellular activity using a reporter vector etc, consider the general tips below.

Transfection reagents for plasmids

If you are transfecting plasmids, a transfection reagent like FuGENE from Promega is a good option as it allows you to prepare your transfection mix in normal cell media rather than having to use a special transfection media.

Transfection reagents for siRNA / miRNA

If your transfection products are much smaller than plasmids (e.g. microRNA mimics, siRNA) a transfection reagent like Lipofectamine RNAiMAX from Thermo Fisher Scientific is a good option.

Transfection optimisation

Performing a series of transfections, with different transfection mix concentrations and different harvest times will allow you to determine the ideal transfection conditions. Taking the time to optimise your transfection conditions will give you better results once you commence your main experiments.

Plate sizes for protein & RNA

Choose your plate size carefully based on whether you are harvesting RNA or protein. If you are harvesting RNA you will be able to use a smaller well size (e.g. 12 well plate) which means you will have to grow up less cells and use less reagents. If you are harvesting protein, a 6 well plate is commonly used to obtain a reasonable amount of protein.

Treat in triplicate

Each time you perform a transfection experiment, make sure to perform each treatment in triplicate. When performing triplicates if two wells give a similar result whilst the third well gives a different result, this will allow you to identify when something has gone wrong. Triplicates will also allow you to calculate means and other statistical analyses for each experiment. This will give you reliable numbers to compare when repeating the entire experiment. (Three independent experiments are recommended for the biosciences and are usually required for publication).