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

Electrophoresis. 2006 Jul;27(14):2865-8. doi: 10.1002/elps.200500568.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / genetics
  • Carbocyanines / chemistry
  • Cells, Cultured
  • DNA Primers / chemistry
  • DNA, Single-Stranded / analysis
  • DNA, Single-Stranded / chemistry
  • Fluorescent Dyes / chemistry
  • Glutathione Peroxidase / genetics
  • Humans
  • Organic Chemicals / chemistry*
  • RNA, Messenger / analysis*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*

Substances

  • Actins
  • Carbocyanines
  • DNA Primers
  • DNA, Single-Stranded
  • Fluorescent Dyes
  • Organic Chemicals
  • RNA, Messenger
  • SYBR Gold nucleic acid gel stain
  • cyanine dye 5
  • Glutathione Peroxidase