Correlation of mRNA delivery timing and protein expression in lipid-based transfection

A. Reiser; D. Woschée; N. Mehrotra; R. Krzyszton; H. H. Strey; J. O. Rädler

The lipoplex solution was made by mixing Lipofectamine TM 2000 with mRNA like previously described (18) . Equal fractions of this lipoplex solution were diluted to the same final mRNA concentration of 0.5 ng/µl with L15 medium containing different FBS fractions and incubated for further 5 min prior transfection. We performed the transfection during the time-lapse measurement using a tailored tubing system that was connected to the channel slide 4 h after cell seeding. First, all channels were rinsed with 37°C warm PBS. Afterwards, each nanocarrier solution was added to one channel of the single-cell array to measure the protein expression dynamics dependent on FBS fraction. After 1 h of incubation with the transfection complexes all channels were washed with L15 medium supplemented with 10% FBS, which remains in the channels for the remaining measurement. After the addition of the mRNA complexes we measured the cells for at least 15 h with a 10 min time resolution to observe the translation kinetic for each successfully transfected cell.