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.

Answer

There are reasons that can influence expression after transfection, but before troubleshooting all the possibilities, a transfection experiment with a positive control reporter mRNA and the new Lipofectamine® MessegnerMAX™ mRNA transfection reagent could be the solution. If this does not yield good results it might be best to try an alternative delivery solution or different cells. However, if this gives acceptable results, the next step would be to try the mRNA of interest with the MessengerMAX™ reagent. If there are expression level concerns at this point, it might be the mRNA that is being used and troubleshooting from this perspective might be needed. For example, some questions to ask would be: Is there a 5’ cap? Is there a poly(a) tail? Is the mRNA pure? Do I get a single band on a gel? Was the DNA template clean? There is information available to help answer many of these questions and more – click here.