We recently finished our Ask the Expert discussion on Got mRNA?  Solve DNA transfection issues with mRNA transfection.This week we had many interesting questions and helpful suggestions on topics including mRNA trasfection advantages, speed, media and troubleshooting expression. In addition, feedback was provided on addressing challenges like toxicity, variation, and transfection in low pH.

Typically, DNA enters the nucleus when cells divide because cell division creates small nuclear pores.  In non-dividing primary cells DNA doesn’t enter the nucleus making these cells very hard-to-transfect. If DNA entry is a bottleneck, why not deliver mRNA directly?

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 – Got mRNA?  Solve DNA transfection issues with mRNA transfection

Question:

I have been getting some toxicity in my mRNA transfections. What would you recommend for minimizing toxicity?

The Answer:

We also observed some toxicity in our initial experiments with mRNA. However, incorporating the proper 5’ UTR and 3’ UTR sequences into the template used for in vitro transcription quickly resolved it. Another key factor was the purity of the mRNA. Ensure that the 260/280 OD ratio is between 1.8 and 2.1. The quality of the mRNA can also be determined by running a small sample on a gel to check for the proper size. In some scenarios, it might be best to also incorporate chemically modified nucleotides to the transcription reaction. Another reason for toxicity may be a result of the cells themselves; if the cells are at too low a density then there will be significant toxicity (ideal viable cell density on the day of transfection is between 70-90% confluence).

Question:

We have seen quite a bit of variation in our transfection efficiency based on the number of passages prior to transfection. Do you have advice on the best passage number?

The Answer:

We have seen this as well throughout our labs. Since we identified this issue, we have implemented a standard best practice of only utilizing cells for transfection between 5- 20 passages because at a low passage there is very low transfection efficiency and at a higher passage the optimal dose of transfection shifts. We have also implemented the standard use of a positive control within each transfection experiment to quantitatively determine and track efficiency from week-to-week.

Question:

We currently use DNA for transfection, can you tell me what the advantages are of mRNA transfection and it is cell line specific?

The Answer:

There are many advantages to using mRNA vs DNA for transfection and they are cell line specific.

• A much higher level of transfection efficiency

If you are working with a difficult to transfect cell type, where DNA transfection yields less than 30% efficiency, transfecting an mRNA alternative can provide up to 80% transfection efficiency. Part of the hurdles that are encountered with DNA delivery has to do with the multiple steps required during transfection. In order to have a protein expressed in a cell, typically DNA is transfected, enters the nucleus, transcribed to mRNA, exported from the nucleus and translated into a protein in the cytoplasm. When delivering mRNA directly, the mRNA is present in the cytoplasm and ready for immediate translation to protein. However, if you are currently satisfied with the level of transfection achieved in the cell line that you are working with, there is no need to switch to mRNA.

• It is a foot-print free method with no risk of genomic integration

mRNA transfection is transient and does not enter the nucleus or pose a risk of integrating with the cellular host DNA and is currently being researched for possible vaccine replacement and disease model development.

• Additionally, transfection of mRNA with the newly developed Lipofectamine® MessengerMAX™ reagent, provides higher efficiency in a wider range of cell types

(e.g. primary neurons, primary hepatocytes, primary keratinocytes, primary fibroblasts, iPS cells, hNSC, mESC, Raw 264.7, SH-SY-5Y, HT-29). This is a result of its ability to delivery the highest amount of mRNA independent of the cell model being used.