I need some help with deciding the next steps for my experiment and would like to see what everyone has done.

We have developed an mRNA vaccine we would like to test in vitro. I have chosen RAW264.7 cells. The mRNA is encapsulated in LNP and will have the N1-methylpseudouridine modification.

I need to test:

1. The stability of the mRNA

2. The desired protein product is being made (which I suppose simultaneously tests whether or not it is being taken up into the cells

3. Is the mRNA vaccine cytotoxic?

To test the stability:

To test for the desired protein product:

I will transfect the cells with the vaccine, I will not use lipofectamine as they are already encapsulated in LNP which will facilitate their uptake into the cell. My control will be GFP mRNA also encapsulated in LNP

To test the cytotoxicity:

I will use almarBlue after exposing the cells to the mRNA vaccine

Now the questions I have are about specific choices to make:

1. Would my GFP control need to also be modified with N1-methylpseudouridine?

2. In terms of adding a FLAG tag to the mRNA, is the best way to detect it through MS analysis or through immunoblotting? If through targeted MS analysis, would I still need to do a pull down using beads for anti-flag?

3. What's the best way to quantify the uptake of GFP into the cells? Flow cytometry or could it also be run on the mass spec?

4. What is a good amount of mRNA to start with in general for cell viability?

Any papers as well that pretty much do the same thing would be greatly appreciated!

Thank you in advance