We recently purchased eGFP mRNAs (L-6101 and L-6402) as controls for mRNA delivery experiments. I have transfected HEK293 and DU145 cells using lipofectamine 3000 and PEI (5:1 weight ratio) and am unable to see GFP expression (by FACS) after 7 or 24 hours. By FACS (looking at cy5) the delivery looks good. I have tried both the labeled (L-6402) and unlabeled (L-6101) side by side and transfected 100 or 200 ng/well (96-well plate) into cells at about 50% confluency. Control transfections using these cell lines with a CMV-eGFP plasmid produce almost off-scale GFP expression when measured by FACS. Is there something wrong with my protocol and what is the timing and level of GFP expression I should expect in comparison to a plasmid transfection?

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Dear Scott,

Thank you for your inquiry. I would suggest checking for mRNA degradation. Make sure you are using serum free reagents (ie Optimem) that are rigorously RNAse free. We use special pipets for mRNA and for example do not do minipreps or maxipreps with these pipets. RNasezap can be used to clean work surfaces and pipets. FACS signal from the Cy labeled RNA does not ensure that there was good delivery since the RNA could simply be trapped in an endosome. This experiment should work well in the 293 cells, we have no experience with the other cell line. Additionally, you make want to experiment with the timing. Though I would expect to see EGFP expression at your indicated time points, many factors influence expression and half-life. We and others typically see peak expression between 12-18 hours.

Regards,
Brea