title: A new method of competitive reverse transcription polymerase chain reaction with SYBR Gold staining for quantitative analysis of mRNA.
abstract: 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.
authors: Ochanomizu University, Institute of Environmental Science for Human Life, Otsuka, Bunkyo, Tokyo, Japan.
authors: Rieko
authors: R
authors: Oba
authors: Yasuko
authors: Y
authors: Kudo
authors: Naomi
authors: N
authors: Sato
authors: Reiko
authors: R
authors: Noda
authors: Yuzuru
authors: Y
authors: Otsuka