Comparison of the DART and DLR® reporter methods. In separate experiments, HEK293T cells were co-transfected with pMEF3/TATA-PLR1 or pMEF3/TATA-Luc+ in the presence and absence of plasmids expressing Six4 and Eya2. The pCMV-PLG3 or pRL-CMV Lucs were included as the corresponding normalization controls. (a) After normalization to the pCMV-PLG3 or pRL-CMV activity, the fold reporter gene activation of pMEF3/TATA-PLR1 or pMEF3/TATA-Luc+ in the absence of both Six4 and Eya2 was set to 1.0. All reporter signals were expressed as the mean fold-amplification ± STD (duplicate transfections, assayed in triplicate). The normalized reporter activities were not significantly different (t-test) between the two methods when comparing MEF3/TATA alone (P = 0.97), in the presence of Six4 (P = 0.14), and in the presence of Six4 and Eya2 (P = 0.22). (b) The S/N of the reporter methods is represented by the ratio of the raw signals from pMEF3/TATA-PLR1 and pMEF3/TATA-Luc+ compared to those of the corresponding lysed untransfected cells (hatched bars). (c) Plots showing the simultaneous monitoring of the pCMV-PLG3 normalization control (black) and pMEF3/TATA-PLR1 reporter (red) signals using DART (with filters) to produce the normalized data illustrated with red bars in panel a.