The performance of our mRNA has been extensively validated with in vitro cellular experiments. We routinely transfect our mRNA into a wide range of cell types and analyse expression, and can provide useful advice.
A common experiment to validate the expression of BASE mRNA is to transfect into human hepatoma, HepG2 and HUH7 cells. On the day of transfection, cells are seeded in a 96-well plate to achieve the optimal confluency of around 80%.
We typically use MessengerMAX (Invitrogen) which is a simple transfection reaction that we have optimised for in vitro delivery. However, electroporation of mRNA may be preferable depending on the cell type.
Figure. eGFP mRNA expression in mouse embyronic neurons.
Our scientific team has experience in mRNA research, including the development of mRNA drug candidates.
We can provide bespoke research services including;
(i) Validate the translation and expression of your mRNA candidate in HEK293 cells.
(ii) Measure the immunogenicity of your mRNA candidate using a THP-1 activation assay.
For each well in 96-well plate, 0.3μl of MessengerMAX and 50ng of mRNA are first diluted in serum-free OptiMEM media before being combined and added directly to cells. The amount of mRNA and transfection reagent is optimised according to cell-type and required dosage.
After 24 hours, mRNA expression is measured with quantitative fluorescence. Within 4 hours post-transfection, we observe rapid onset of expression, however, the peak of expression usually occurs around 24-hours and then decays over the following days. Reporter mRNA expression can also be measured by using other assays, such as luminescence or western blotting.
We use this approach to routinely validate expression of BASE mRNAs in a diversity of cell types.
Webinar with Dr Seth Cheetham, in partnership with Integrated DNA Technologies (IDT), shows how to quickly DNA templates can be made for synthesis of mRNA for pre-clinical research.