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Transient co-transfection of plasmids is a method that is commonly employed for cellular protein-protein interaction studies, transcription factor studies, and gene knockdown studies using shRNA encoding plasmids. Thus, co-transfection is useful for a broad scientific community. In the present study, X-tremeGENE HP and X-tremeGENE 9 Transfection Reagents (Roche) were tested for their co-transfection efficiency using two autofluorescent reporter genes, enhanced green fluorescent protein (eGFP), and red fluorescent protein (mKate2).
Transfection and automated imagingHeLa Cells (ATCC) were plated at a density of 1.2 x 105 cells/100 µl per 96 well. The cells were transfected with PC3.1eGFP and pmKate2 plasmids (Evrogen) 24 hours after cell seeding. Three different pmKate2/PC3.1eGFP ratios were titrated (0.5 + 1.0, 0.5 + 0.5, 1.0 + 0.5 µg plasmid per 100 µl complex). In addition, four different amounts of X-tremeGENE HP and X-tremeGENE 9 Transfection Reagents, respectively, were tested for complex formation (1, 2, 3, or 4 µl reagent per 100 µl complex). Transfection efficiency was measured 48 hours post transfection using the Cellavista Analyzer and HOECHST 33342 staining.
ResultsTransfection efficiencies with two plasmids vary depending on the different ratios of the plasmids themselves and the relation of whole DNA/transfection reagent (see Figures 1, A, B, and E).
Both X-tremeGENE HP and X-tremeGENE 9 Transfection Reagents showed a high co-efficiency (see Figure 1, C and D).
X-tremeGENE HP Transfection Reagent showed a more consistent transfection efficiency over all ratios in the tested HeLa cells.
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Georg Hannig is research scientist, and Cordula Jany (
