Introduction

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 imaging

HeLa 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.

Results

• Transfection 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.

Figure 1.  Successful co-expression of eGFP and mKate2. (Click to enlarge)

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