Improving the Efficiency and Performance of your Transfection

By on March 18, 2014
Expert Session Summaries

We recently finished our Ask the Expert discussion on transfection optimization for improved efficiency and performance. This week we had many interesting questions and valuable answers and suggestions. Troubleshooting topics involved drops in cell viability, co-transfecting DNA and siRNA, gene expression, antibiotic use, cell line selection, and transfection after thawing. Several cell lines were discussed including hematopoetic cells, HEK cells, primary cells, and cancer cell lines. In addition ideal transfection conditions were explored along with serum in the tranfection media.

Transfection is a common, yet sophisticated method that is frequently used to artificially deliver nucleic acids (DNA or RNA) into cells for a variety of applications. To efficiently introduce nucleic acids to the cell, a chemical method such as lipid based reagents or a physical method such as electroporation is most commonly used. These nucleic acids can alter properties of the cell, allowing for the study of gene function and protein expression within the context of the cell. However, there are a number of important factors, such as reagent dose, nucleic acid dose, cell density, complexation media, incubation time, etc., that can affect the efficiency of transfection. The difference between a good and bad transfection, can ultimately determine how many times an experiment will be repeated. Understanding the interaction between these key factors and the importance of optimization for a particular cell type can help to reduce the cost and consumption of time and reagents.

This Ask the Expert Session was Sponsored by Life Technologies and hosted by Nektaria Andronikou. Nektaria joined Life Technologies in 2010 and is currently working with the transfection team on the development of new delivery methods targeting relevant cellular models that will enable the use of exciting new technologies. She received a Bachelor of Science in Biochemistry with a minor in Cellular and Molecular Biology from UCSD. She began her professional career at ISIS Pharmaceuticals, as a research associate for the Cardiovascular Drug Discovery program, screening numerous pre-clinical targets that led to the discovery of the now FDA approved antisense drug, Kynamro.

Below is a sneak peek of the discussion. For a full transcript of the discussion, please see – Ask the Expert – transfection optimization for improved efficiency and performance.

After transfection, I regularly get a drop in cell viability. Is there any reason why this continues to happen and what things should I be troubleshooting?

The Answer:
You might try removing the media that contains the transfection complex 4-6 hours post transfection and replace it with growing culture media. If this doesn’t help with cell viability, you might need to do a small experiment to optimize a few key parameters that can influence the performance of your transfection. Cell density can be an important factor for cell viability and transfection. If cell density is too low at the time of transfection, then a big drop in viability may be seen. Also, ensure that high quality DNA is used. We suggest to test two different cell densities, two to four recommended lipid doses and two DNA doses.

How can you determine if your cell line will be easy or challenging to transfect? Is there anything you can do to make it easier if you have a difficult line?

The Answer:
Typically, we identify a cell line as “difficult to transfect” by using a positive control reporter, such as GFP or luciferase. Multiple delivery methods should also be tested for any new cell type and the most commonly and easy to use method is lipid mediated; however, if this approach doesn’t yield the desired results, electroporation can be performed using the same positive control plasmid. There are certain mechanisms of why specific cell types are hard to transfect. Some cell types, such as MCF7 or HepG2, prefer to grow in clumps or clusters which is not ideal for transfection because minimal membrane surface is exposed which compromises uptake; there are other cell types, such as blood or immune cells, that lack the proper endocytic machinery, which again can minimize uptake; and there are other cells, such as macrophages, that have an evolved uptake mechanism, but quickly breakdown and destroy endosomal contents.

The following steps can help with optimizing transfection for lipid mediated delivery with DNA or RNA, to improve the efficiency for a wide variety of cell types, even the difficult to transfect ones:

  • Performing the recommended reagent protocol, with the indicated doses, will help to determine the “sweet spot” of the reagent for a particular cell type
  • Optimizing for cell density so that the cells are 70-90% confluent on the day of transfection, can help to improve efficiency by almost 10-15%
  • Maintaining healthy cells in log phase during sub-culturing
  • Controlling cell passage number post thawing is important for the health of the cells; most cell types should be used between 4 and 25 passages for optimal transfection
  • Using the recommended cell culture media

Can you describe the ideal cell culture conditions for the best transfection results and does serum in the media have any impact on transfection success?

The Answer:
The ideal cells are between passages 5-20 after thawing and growing in log phase. Prior to transfection, cell viability should be greater than 90% and 70~80% confluent. For transfection, we recommend using Opti-MEM (serum-free) for lipid/payload complex formation and culture medium supplemented with 10% serum. Lower amount of serum (<10%) works as well but post-transfection (4-24 hr) media change with complete growth media is recommended.


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