First we determined the luciferase expression levels of the firefly (FNP) and Gaussia (GNP) luciferase reporter constructs when transfected alone or in combination by using the transfection assay. As shown in Figure 2 , single transfection of each reporter gene resulted in high expression levels of both the firefly ( Fig. 2A) and Gaussia (Fig. 2B ) luciferase genes. However, when the reporter constructs were co-transfected, the firefly luciferase expression level was dramatically reduced ( Fig. 2A) , while the Gaussia luciferase expression level in the same cells was not affected when compared to the single-transfected cells (Fig. 2B) . The differential expression of firefly and Gaussia reporter plasmids when transfected alone or together can also be illustrated by plotting the normalized ratio of firefly to Gaussia luciferase activity (normFluc/ Gluc; the normalized ratio's were calculated as indicated in the Materials and Methods section). As shown in Figure 2C , this ratio is significantly decreased upon co-transfection of the two reporter constructs, when compared to the ratio of the luciferase expression levels in the single-transfected cells. Similar results were obtained at earlier and later time points post transfection (data not shown). This indicates that the balance of firefly and Gaussia luciferase expression is strongly in favor of Gaussia luciferase, when both reporter constructs are present within the same cell. Thus, the results indicate that expression of the firefly luciferase gene is Figure 2 . Competition between firefly and Gaussia luciferase reporter genome segments. Plasmids encoding firefly (FNP) or Gaussia (GNP) luciferase reporter constructs were transfected alone (Single) or in combination (Co). Luciferase expression was induced by simultaneous cotransfection of polymerase and NP expression plasmids (transfection assay). A) Firefly luciferase activity after transfection of FNP or FNP together with GNP. B) Gaussia luciferase activity after transfection of GNP or GNP together with FNP. C) Normalized ratio of firefly to Gaussia luciferase activity (Fluc/ Gluc) when FNP and GNP were transfected singly or in combination. D) Quantitative RT-PCR analysis of mRNA levels derived from FNP and GNP after single or co-transfection of these constructs. RNAs were extracted 24 h post-transfection and subjected to quantitative RT-PCR. The comparative Ct method was used to determine the relative mRNA levels using the housekeeping gene GAPDH as a reference. The mRNA levels were normalized relative to the samples in which a single reporter construct was transfected. E) Normalized ratio of firefly to Gaussia luciferase activity (Fluc/Gluc) when reporter gene constructs FNP and GNP were transfected singly or in combination. The amounts of reporter gene constructs transfected are indicated. Significant differences in A-D are indicated (**; P,0,01). doi:10.1371/journal.pone.0047529.g002 negatively affected by co-transfection of the Gaussia luciferase reporter plasmid.