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A new method of competitive reverse transcription polymerase chain reaction with SYBR Gold staining for quantitative analysis of mRNA

✍ Scribed by Rieko Oba; Yasuko Kudo; Naomi Sato; Reiko Noda; Yuzuru Otsuka

Publisher
John Wiley and Sons
Year
2006
Tongue
English
Weight
197 KB
Volume
27
Category
Article
ISSN
0173-0835

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✦ Synopsis

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 β‐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.

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