mRNA transfection

We have used Thermo Lipofectamine MessengerMax and Milenyibiotec StemMACS reagents with good success in both immortalized cell lines and primary cells.

For each, we adapted the protocol from the manufacturer’s protocol.

MessengerMax transfection protocol

  1. Seed cells to be 70-90% confluent at time of transfection.

Note

Amounts given are for transfecting a 96-well with 100 ng of mRNA. If you would like to transfect more mRNA, adjust the amount of MessengerMax (2:1 ratio of reagent to mRNA, i.e., .2 µL MessengerMax per 100 ng mRNA ) Scale up amounts of mRNA, reagent, and KO-DMEM solutions 5x for a 24-well (25 µL total volume) and 12.5x (125 µL total volume) for a 6-well.

  1. Dilute .2 µL MessengerMax Reagent in 4.8 µL KO-DMEM, mix well, and incubate 10 minutes at room temperature.

  2. Dilute 100 ng mRNA in KO-DMEM to a total volume of 5 µL, and add to the MessengerMax/KO-DMEM mixture.

  3. Incubate for 5 minutes at room temperature.

  4. Add the 10 µL of mRNA-lipid complex to desired 96 well.

StemMACS transfection protocol

  1. Seed cells to be 70% confluent at time of transfection.

Note

Amounts given are for transfecting a 96-well with 100 ng of mRNA. If you would like to transfect more mRNA, adjust the amount of StemMACS (3:1 ratio of reagent to mRNA, i.e., .3 µL StemMACS per 100 ng mRNA ) Scale up amounts of mRNA, reagent, and KO-DMEM solutions 5x for a 24-well (25 µL total volume) and 10x (100 µL total volume) for a 6-well (their recommendations are different from MessengerMax)

  1. Add .3 µL StemMACS to 4.7 µL of StemMACS Transfection Buffer (in 4 C TC fridge) and mix well.

  2. Dilute 100 ng of mRNA in StemMACS Transfection Buffer to a total volume of 5 µL.

  3. Add the diluted StemMACS reagent/buffer solution to the diluted mRNA and incubate for 20 minutes at room temperature.

  4. Add the 10 µL of mRNA-lipid complex to desired 96 well.

Note

If performing multiple mRNA transfections, media change 4 hours post-transfection to reduce cytotoxicity.