A systematic comparison of in-house prepared transfection reagents in the delivery of mRNA or DNA to a wide range of cultured cells.

Transfection is a fundamental molecular biology technique, enabling gene editing, protein expression, and vaccine development. However, transfection efficiency and cytotoxicity vary widely between reagent and cell type, necessitating optimization. Commercial reagents such as Lipofectamine 2000 and FuGENE HD are widely used for their high efficiency, but they are expensive and the efficiency can associate with cytotoxicity. In-house alternatives such as linear polyethylenimine (PEI; 25 kDa and 40 kDa) and cationic lipids-1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-combined with dioleoylphosphatidylethanolamine (DOPE) offer cost-effective options, but their performance across diverse cell types and nucleic acid type (RNA or DNA) remains insufficiently characterized. This motivated us to systematically evaluate transfection efficiency, cytotoxicity, and complex stability of these in-house reagents (DOPE:DOTAP and DOPE:DOTMA tested at molar ratios of 0.5:1, 1:1, and 2:1) over a broad range of reagent to nucleic acid ratios, using plasmid DNA and mRNA encoding mCherry. We performed transfections across 14 cell lines derived from human, monkey, frog, snake, and rodent tissues. We utilized automated fluorescence microscopy for quantifying transfection efficiency, luminescence-based viability assays for cytotoxicity, and studied complex stability during storage at 4°C (0, 4, and 24 hours) through transfection. Results revealed cell line-dependent differences in transfection efficiency, and showed in-house cationic lipid formulations to have a high mRNA transfection efficiency with low cytotoxicity. Lipofectamine 2000 and PEI 40k formed the most stable DNA complexes, but with higher cytotoxicity. This study provides a comprehensive reference for selecting customizable, cost-effective transfection reagents for specific cell and nucleic acid types.