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.
Subject(s)
Organic Chemicals/chemistry , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Actins/genetics , Carbocyanines/chemistry , Cells, Cultured , DNA Primers/chemistry , DNA, Single-Stranded/analysis , DNA, Single-Stranded/chemistry , Fluorescent Dyes/chemistry , Glutathione Peroxidase/genetics , HumansABSTRACT
High level of vitamin B(12) was incorporated into kaiware daikon (Japanese radish sprout) by soaking its seeds in B(12) solutions. Vitamin B(12) amount incorporated into kaiware daikon increased up to 1.5 microg/g wet sprout with the soaking time of seeds in 0-200 microg/ml B(12) solution. Vitamin B(12) could be extracted more from the sample heated for a short time than from that of control without heat treatment.
