ABSTRACT
To clarify the effects of O(2) concentration (cO(2)) on antioxidant gene expression in human hepatocytes, mRNA expression of HepG2 cells cultured at 1, 3, and 5% cO(2) and atmospheric gas-phase, was measured. The expression of some genes fluctuated depending on the cO(2) in the incubator. This indicates that cO(2) is a critically important factor in the investigation of human biological mechanisms.
Subject(s)
Antioxidants/metabolism , Gene Expression Regulation/drug effects , Oxygen/pharmacology , Dose-Response Relationship, Drug , Hep G2 Cells , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
There are several methods available to analyze the mRNA concentration quantitatively. Among them, the competitive reverse transcription (RT-)PCR method is very useful. For this method, Cy5-labeled primers were used, and after gel electrophoresis in 7 M urea, the Cy5-labeled single-strand DNA was measured by a fluorescence detector. However, as the equipment to measure the Cy5-labeled fluorescence is expensive, we developed a new method using SYBR Gold staining. After gel electrophoresis in 7 M urea, the single-strand PCR product DNA was stained with SYBR Gold, and photographed with a standard UV-transilluminator and a standard digital camera with a specific filter for SYBR Gold staining. The photographic image was digitized by an imaging software. We measured beta-actin and plasma glutathione peroxidase (Gpx3) mRNA concentrations of HepG2 cell cultured at 5 and 20% oxygen tension. The Gpx3 expression was increased by hypoxia. The result was equivalent to the data obtained by the real-time PCR analysis.
