Transfection Optimization for Improved Efficiency and Performance


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.

Question 1

Hello, I’m working with multiple cancer cell lines. Can I use the same amount of any transfection reagent for different cells lines?

Great question. The answer is no. Transfection efficiency depends highly on the amount of reagent used per well and maybe very different between reagents. Usually, the protocol that is supplied with the product will provide an optimal range of transfection reagent to use. You can go to to get specific, detailed transfection protocol for the cell lines that you are interested in. If you are still not achieving the performance you desire in your particular cell line, further optimization may be necessary.

Question 2

Hello, I recently started a new project that requires transfection for a few cancer cell lines. I wonder if you could suggest some of the key considerations for successful transfection.

That’s a great question! Successful transfection is influenced by many factors—the choice of the transfection method, viability of the cells prior to transfection, number of passages, degree of confluency, quality and quantity of the nucleic acid used, the amount of reagent can all play a part in the outcome of your transfection experiments. YYou can visit our Transfection page and go over the section ‘Find the Right Transfection Reagent’ to get more detailed information on cell lines that you work with.

Question 3

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 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.

Question 4

What is the best time to transfect cells after thawing them for best results?

The best time to perform transfection after thawing depends on the cell type. A wide variety of immortalized cell lines have optimal performance after passage 4-5 and until passage 20-25. If transfection is performed on low passage cells, post thawing, lower transfection performance will be observed; while cells that are maintained for higher passage numbers, will have an inaccurate and misleading high transfection efficiency. We are also aware of one cell line, NIH 3T3, that has better transfection at a higher passage number, from 10-40. There are also cell lines, such as primary endothelial cells, primary smooth muscle cells, primary corneal epithelial cells that have a much smaller passage window for optimal transfection performance. Since passage number can affect the growth rate of the cells, which can directly affect transfection, we suggest to test the range of recommended reagent doses, which can be found in the revised protocols on our website.

Question 5

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?

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

Question 6

Is it ok to use antibiotics during transfection?

Yes, antibiotics can be used during transfection with any of the Life Technologies lipid based reagents. However, the critical steps of the protocol need to be performed with the recommended OptiMEM Media and not culture media that contains any serum or antibiotics. The transfection protocol involves a two tube protocol and It is important to add the lipid reagent to OptiMEM in one tube and the DNA or RNA to OptiMEM in another tube and mix each tube thoroughly. Then, equal amounts from each tube should be mixed to form the transfection complex and incubated for five minutes at room temperature. The lipid-DNA or lipid-RNA complex can then be added directly to cells in complete culture media. Analysis of transfection results can be performed 24-48 hours post transfection.

Question 7

In primary cells how long should I wait after transfection before I can assay for gene expression?

For delivery in primary cells or any cell model, gene expression is dependent on the downstream assay required for the experiment. A typical window or range is anywhere from as early as 12 hours for expression from mRNA delivery, 24-48 hours for evaluation of expression of a fluorescent protein, to 72-96 hours for evaluation of genome editing tools like TALs or CRISPR. However, there are factors that could influence the efficiency of expression such as size of the DNA or mRNA, the promoter for the DNA, age and health of the cells, the half-life of the protein being expressed, etc..

On another note, a little tip we can offer, if you are trying to improve delivery in primary cells, would be to try an mRNA version of your gene of interest. The reasoning is that mRNA only requires entry to the cytoplasm of the cell as opposed to the nuclear localization that is required for DNA; and since primary cells tend to be in a post-mitotic state, with minimal nuclear envelope disruption, delivery to the nucleus has proven to be quite challenging. Life Technologies offers multiple in vitro transcription kits that are complete with the necessary items to make mRNA in the lab; an ARCA cap, the nucleotides and a RNA specific polymerase are included in the kit. The only requirement is a linearized vector or PCR product of the gene of interest containing a T7 or SP6 promoter. More information can be found on the Life Technologies website.

Question 8

I am trying to co-transfect DNA and siRNA into HEK cells. Can you give me some advice on how the most effective way to do this.

For getting successful co-transfection, you need high quality DNA, a validated siRNA against the gene of interest and log phase growing HEK293 cells. The method or application for co-transfection is important to help you determine the order of delivery and the right reagent for transfection:

1. To study the effect of siRNA knockdown of the gene that is expressed by the co-transfected plasmid, transfection can be performed at same time by using one reagent, such as Lipofectamine® 3000 or Lipofectamine® 2000.
2. To study the effect of siRNA knockdown of an endogenous gene, siRNA transfection should be done first using an RNAi specific delivery reagent such as Lipofectamine® RNAiMax. 4-48 hours after siRNA has been delivered, DNA can be transfected using Lipofectamine® 3000 or Lipofectamine® 2000. The time of post-transfection delivery of DNA may need to be optimized.

Once the appropriate method of delivery is determined based on the application, transfection should be optimized for DNA, siRNA and lipid delivery reagent doses. For transfecting HEK293 in 96-well, we typically use these amounts per well:
• DNA: 0.1-0.2 ug
• siRNA: 1-3 pmoles
• Lipid reagent: 0.1-0.3 ul

Question 9

How do I address the incredible difficulty of transfecting hematopoetic cells, specifically immune system cells (e.g., macrophages such as J774A.1 and THP-1)?

Do you really need to use this cell type?

These notoriously difficult cells require a certain amount preparation, because a variety of delivery techniques might need to be tested for success. If you are trying to over express a gene with DNA delivery, the newly developed Lipofectamine® 3000 can be tried first for the simplicity of using a lipid based delivery method. However, if this method doesn’t yield any results, the Neon™ Electroporation system with DNA is recommended. You could also try to do electroporation with an mRNA transcript of your gene of interest, which only needs to be delivered to the cytoplasm for protein translation. If all these previous methods fail, a virus can be used for gene expression.

If gene silencing with siRNA is desired, the lipid based Lipofectamine® RNAiMax can be tested; however, the Neon™ Electroporation system is the recommended method. If this proves unsuccessful, an shRNA vector can be delivered.

Question 10

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?

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.

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