mRNA Transfection Toxicity (Lipid Transfection)

Question

Accepted Answer

Cell toxicity observed during mRNA transfection experiments with MessengerMAX reagent Common causes: 1) Improper 5' UTR and 3' UTR sequences in template 2) Poor mRNA purity (OD 260/280 not between 1.8-2.1) 3) Low cell density at transfection (should be 70-90% confluence) 4) Missing chemically modified nucleotides in transcription 5) High passage number of cells affecting sensitivity Solutions: 1) Incorporate proper 5' UTR and 3' UTR sequences into transcription template 2) Ensure mRNA OD 260/280 ratio between 1.8 and 2.1 3) Run mRNA on gel to check proper size and quality 4) Maintain viable cell density between 70-90% confluence at transfection 5) Consider incorporating chemically modified nucleotides in transcription 6) Use cells between 5-20 passages only 7) Include positive control reporter in each experiment