A quantitative RT-PCR assay to monitor luciferase reporter mRNA levels
Bioluminescent reporter assays are critical for functionally characterizing gene regulatory elements. Despite the wide-spread usage of reporter assays, the accurate interpretation often requires additional measurements beyond reporter enzyme activity. For example, when studying cis-acting RNA elements involved in post-transcripitional control it is critical to measure the steady state reporter mRNA levels. This can assist in drawing inferences about the underlying mechanism responsible for any differential reporter activity. My thesis work describes development of an RT-qPCR assay to monitor mRNA levels from a transiently transfected dual luciferase reporter system. After validating individual qPCR primer sets, this assay was applied to a comparative transcriptomics study, which previously identified orthologous mRNA isoforms with different polyribosome association profiles. It was shown that a single nucleotide variant in the GGCX and MELK orthologs of humans and chimpanzees was sufficient to modulate luciferase enzyme activity of reporters harboring single exons from these genes. The RT-qPCR results demonstrate that the reporter mRNA levels were not affected by the SNV, indicating that differential reporter activity is due to the translational control of orthologous exons.