How much mRNA is needed to transfect cell cultures in 24-well plates?

There are typically two avenues for transfection of mRNA depending on cell type:

1. For adherent cells, use a transfection reagent such as MessengerMax (Invitrogen), mRNA TransIT (Mirus) or mRNA-In (MTI-GlobalStem).

a. For a 24-well plate format, use a ratio of 500 ng mRNA:1 ul transfection reagent in a total volume of 50 ul complexed mRNA/lipid per well. This is a good starting point and is described in this protocol. Diluting your working stock of mRNA down to 100 ng/ul works well to establish manageable volumes for mastermixes.

b. We’ve only used TransIT and mRNA-In in-house – from our experience these reagents give a dose response of FLuc activity from between 100 ng to 400 ng. Therefore, a minimum starting amount of 100 ng is sufficient. Use 100 ng mRNA: 1ul reagent in a complexed final volume of 50 ul per well.

c. Ratios for other plate formats need to be optimized according to the manufacturer’s instructions.

2. For cells in suspension, such as CD34+ cells, electroporation has traditionally been the mode of mRNA delivery. However, transfection reagents have advanced and some are able to transfect cells in suspension reliably, such as the transfection reagent mRNA-In (MTI-GlobalStem).

a. For electroporation, an instrument from Lonza is recommended based on advice from a trusted collaborator.

b. Use 1-15 ug of mRNA per million cells in a 100 ul volume.