Figure 3. Induction of PGK-1-HRE luciferase reporter by hypoxia using the split transfection protocol.

In the split transfection luciferase assay, cells are transfected with an experimental luciferase reporter alone. Post-transfection, the plate is split equally into multiple plates as required. Differences in transfection efficiency are accounted for by splitting out the original plate at equal cell number, thus eliminating the need for normalization using a constitutive reporter. Experimental reporter luminescence under test conditions can then be directly compared with that of the control condition to obtain results as fold change over control. Rcho-1 trophoblast cells were plated at equal cell number of 1.0 × 10⁵ cells and transfected with PGK-1 and then treated with the indicated level of oxygen for 18hrs. Cells were collected and PGK-1-HRE reporter activation was analyzed. Results are the average of three independent experiments. Luciferase activity at each concentration was analyzed and is shown relative to activity at 21% oxygen. Error bars represent standard deviation. Significance was denoted as *p≤0.01 or **p≤0.001 and was determined using a one way Anova with Tukeys post hoc test.