Analysis of human CYP2E1 mRNA in a HepG2 cell line by reverse transcription-polymerase chain reaction (RT-PCR)

grantor: University of TorontoThe research interest of this thesis work focuses on the regulation of human CYP2E1 mRNA in the absence and presence of potential endogenous inducers, cAMP and insulin. The study of human CYP2E1 mRNA stability and the effects of endogenous inducers on the levels of mRNA has been hampered by the inability to establish a cell culture model system that yields consistently detectable levels of CYP2E1 mRNA. In this study we have reassessed HepG2 cells as a model for human CYP2E1 mRNA expression. In these cells, we (and others) have demonstrated the lack of CYP2E1 mRNA detection by Northern analysis. We have developed a reverse transcription polymerase chain reaction (RT-PCR) procedure that facilitates effective detection of CYP2E1 mRNA. This study details the optimization of RT-PCR parameters for CYP2E1 and GAPDH mRNAs. As validation of the use of RT-PCR as a method for the analysis of mRNA stability, we assessed the expression of GAPDH mRNA from HepG2 cells treated with transcriptional inhibitor, 5,6-dichloro-1 $\beta$-D-ribofuranosylbenzimidazole (DRB), by Northern and RT-PCR methods. The amount of GAPDH mRNA present at increasing times after DRB exposure is reflective of mRNA stability. (Abstract shortened by UMI.)M.Sc