mRNA transfection
=================
We have used `Thermo Lipofectamine MessengerMax <https://www.thermofisher.com/order/catalog/product/LMRNA001?gclid=Cj0KCQiAm5ycBhCXARIsAPldzoUQKASct6CpctkEyzxfPpObolE2SZKRp_dj1xPJys2Td34cCGrxLcwaAseqEALw_wcB&ef_id=Cj0KCQiAm5ycBhCXARIsAPldzoUQKASct6CpctkEyzxfPpObolE2SZKRp_dj1xPJys2Td34cCGrxLcwaAseqEALw_wcB:G:s&s_kwcid=AL!3652!3!535167329917!!!g!!!398236708!127976663191&cid=bid_clb_tfx_r01_co_cp0000_pjt0000_bid00000_0se_gaw_dy_pur_con&s_kwcid=AL!3652!3!535167329917!!!g!!>`_ and `Milenyibiotec StemMACS <https://www.miltenyibiotec.com/US-en/products/stemmacs-mrna-transfection-kit.html#130-132-949>`_ 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 opti-mem, mix well, and incubate 10 minutes at room temperature.
2. Dilute 100 ng mRNA in opti-mem to a total volume of 5 µL, and add to the MessengerMax/opti-mem mixture.
3. Incubate for 5 minutes at room temperature.
4. Add the 10 µL of mRNA-lipid complex to desired 96 well. 

.. note:: KO-DMEM can also be used instead of opti-mem media. KO DMEM will have lower efficiency.

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.