Dear all,
My lab is currently synthesising mCherry-mRNA by T7 RNA polymerase, Vaccinia Capping system, 2' O-methyltransferase and E. coli poly(A) polymerase kits from the New England Biolab. I did the Nanodrop to determine the product concentration between each step and everything looks alright. However, when I tried to do the transfection by using PEI 25k Da (250ug/ml) in different N/P ratio on Hek293T, the mCherry signal was very dim, compared to the mCherry plasmid, even 2ug mRNA with 5ul PEI was applied to 50,000 cells.
So the question is, are there any ways to improve the protein expression in the mRNA transfection. Moreover, after the expression, are there any standards to evaluate the quality of protein expression (i.e. what is the positive control for the mRNA expression?).
Also, any nice transfection reagents suggest for the RNA transdution?
Many thanks and have a nice day
Andy
Hey Andy and a happy new year,
We recently established a method for mRNA transfection with the JetMessenger transfection reagent and in vitro transcribed and immunologically silenced mRNA. This method was used for in vitro transfection of ex vivo (peritoneal macrophages), in vitro (bone marrow-derived macrophages) and MEF cells so far. But you can give it a try also for your cells, if you want.
We reached transfection efficiancies of 80 - 90 % with MEFs and BMDM and 65-70% with PMs. The JetMessenger Kit is designed especially successful for mRNA transfection. So I think, your transfection reagent is the problem.
Your mCherry mRNA is the right control. If you get increasing fluorescence in your cells after transfection, then the transfection itself works (which does not mean your contruct of choice works). If you want to be sure, that your protein is build, you have to run a Western Blot in your transfected cells.
If you want to check your mRNA quality, you can run an denaturating RNA gel to see if your mRNA has the right size. The Nanodrop only tells you that there are RNA nucleotides in your solution but not, if it is a continous mRNA strand of the right size.
For a detailed protocol (including primer desing, kits of choice and purification of mRNA, and control gels) please have a look at:
Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA. J. Vis. Exp. (Pending Publication), e60143, In-press (2019).
All the best and stay healthy,
Marc
Hey Andy and a happy new year,
We recently established a method for mRNA transfection with the JetMessenger transfection reagent and in vitro transcribed and immunologically silenced mRNA. This method was used for in vitro transfection of ex vivo (peritoneal macrophages), in vitro (bone marrow-derived macrophages) and MEF cells so far. But you can give it a try also for your cells, if you want.
We reached transfection efficiancies of 80 - 90 % with MEFs and BMDM and 65-70% with PMs. The JetMessenger Kit is designed especially successful for mRNA transfection. So I think, your transfection reagent is the problem.
Your mCherry mRNA is the right control. If you get increasing fluorescence in your cells after transfection, then the transfection itself works (which does not mean your contruct of choice works). If you want to be sure, that your protein is build, you have to run a Western Blot in your transfected cells.
If you want to check your mRNA quality, you can run an denaturating RNA gel to see if your mRNA has the right size. The Nanodrop only tells you that there are RNA nucleotides in your solution but not, if it is a continous mRNA strand of the right size.
For a detailed protocol (including primer desing, kits of choice and purification of mRNA, and control gels) please have a look at:
Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA. J. Vis. Exp. (Pending Publication), e60143, In-press (2019).
All the best and stay healthy,
Marc
I agree with Marc Herb : nanodrop is not informative here, while a gel absolutely would be.
Also be aware that "naked mRNA" is not necessarily handled the same way as mRNA produced within a cell: even with polyA tailing and capping, your in vitro mRNA will lack all the accessory proteins that typically accrete around a message during synthesis and processing (cellular mRNA is better thought of as ribonuclear protein complexes, or mRNP). Cells very rarely encounter completely naked mRNA, and so when they do, it is not uncommon for this to result in
None of this applies to plasmids, where the mRNA is produced inside the cell, using cellular machinery, and is thus handled in a more conventional manner.
This is absolutley right and for the cell type used by Andy usable and correct. We mainly work with macrophages and, as immune cells, DNA in the cytosol, especially in form of a plasmid, leads to cell death and we did not observe nay transcription from a plasmid (not even GFP) in macrophages. So we were forced to switch to mRNA. Additonaly, I am not talking about RAW cells- These are cancer cells, which some capabilites of a macrophage and these cells are also transfectable with plasmids. For all other cell types I would also use plasmids for the reasons John Hildyard mentioned above.
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