Factors affecting sensitivity of dual luciferase reporter assay for detection of gene regulation in 293T cells

Abstract

The Luciferase reporter gene is one of the most popular methods for studying gene expression and regulation because of its convenience, simple and the broad dynamic range of applications. However, a limitation of this method is a high variability of measurement affecting consistency and sensitivity leading to varied statistical significances. Here, we investigated the factors affecting the sensitivity and consistency of detection of posttranscriptional regulatory sequences in the dual luciferase reporter system. We generated firefly luciferase reporter plasmids containing 3’untranslated region (UTR) of a gene. One construct contained a wild type and 2 constructs contained mutant binding sites for miRNAs. Each plasmid construct was diluted to the concentration of 500 ng, 50 ng and 25 ng and was co-transfected with the renilla reporter plasmid to 293T cells using the ratio between firefly/renilla plasmids of 10:1. Then cells were measured for dual luciferase activities.  We also compared luciferase activities regarding the number of mutation sites to assess whether they affected the detection of luciferase sensitivity.  Our results indicated that at the 50 ng concentration of the reporter plasmid gave the highest sensitivity and consistency for evaluation of luciferase expression via 3’UTR regulatory region with the highest statistical significance when compared to those of wild type and mutant sequences. Moreover, the numbers of mutation sites also had the effect on sensitivity of the luciferase reporter assay. Thus, optimization of plasmid DNA concentration is essential to improve the sensitivity. This information would be useful for researchers and scientists who employ the dual luciferase reporter system in investigating the posttranscriptional regulation of sequences.