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

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Accepted 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).

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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?

I am trying to troubleshoot our process. Could you tell me some reasons why we may not be seeing expression after transfection? We are using primary cells, so I am not sure if it is due to the difficulty in transfecting these cells or another cause.

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