The transfection efficiency for each plasmid preparation was determined by exposing 7.5 x 106 endotoxin-sensitive RAW 264.7 cells4 to calcium phosphate precipitates containing 15 g of StrataPrep kit, AEC-, MB-, and CsCl-purified pA3-3.4Luc (IL-1Ra promoter/luciferase reporter) plasmid. Luciferase expression in this plasmid is stimulated by endotoxin. Cells were exposed to plasmid-calcium-phosphate precipitates for 5 hours, shocked in glycerol for 90 seconds, and washed. Cells from each plasmid transfection were divided equally into a set of six plates and returned to the incubator.
Forty hours post-transfection, three plates of each plasmid preparation set were treated with an excess (1 g/ml) of E. coli lipopolysaccharide (LPS), while the remaining three plates of each set were not stimulated. After an additional 8 hours of incubation, LPS-stimulated and unstimulated cells for each plasmid preparation were harvested, lysed, and assayed for luciferase activity and total protein. Relative light units (RLU) were normalized to protein concentration and expressed as RLU/mg of protein.
The transfection efficiency of StrataPrep-purified DNA, measured as total
RLU/mg of protein for stimulated cells, was 1.3-, 3.4-, and 2340-fold
higher than stimulated cells transfected with CsCl, AEC, and MB DNA, respectively
1). The fold RLU increase over background (RLU LPS-stimulated divided
by RLU LPS-unstimulated control) for StrataPrep Kit-, CsCl-, AEC-, and
MB-purified plasmid was 29, 18, 12, and 0.16, respectively. The
high induction ratio for StrataPrep DNA reflects both a high transfection
efficiency (indicated by enhanced LPS stimulation compared with the other
preps) and negligible degree of endotoxin contamination, evident from
the low LPS-unstimulated bac