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Marine Biotechnology

, Volume 3, Issue 1, pp 16–21 | Cite as

Quantitative TaqMan PCR Without a Real-Time Thermal Cycler: An Assay for Fish Insulin-like Growth Factor I Messenger RNA

  • Anthony  Dyer
  • Kathleen  Soole
  • George  Matsumoto

Abstract:

The reverse transcriptase–polymerase chain reaction, more commonly known as RT-PCR, has become a widely used tool in molecular biology and is now frequently used in monitoring gene expression levels. A number of variations in the RT-PCR technique now exist including TaqMan PCR (5′ nuclease assay), which is a useful nonisotopic detection method for the quantification of PCR products. To monitor the formation of these fluorescent amplification products a ``real-time'' thermal cycler is normally required. In this study, repeated scanning of PCR products in a 96-well plate format showed that a conventional fluorescent plate reader can be used to generate similar results. To demonstrate the power of this approach, the nutritional regulation of insulin-like growth factor I (IGF-I) was investigated in a marine finfish, the snapper (Pagrus auratus). Hepatic IGF-I messenger RNA levels were shown to significantly decrease after 2 weeks of fasting and returned to fed control levels on refeeding. These results demonstrated that a real-time PCR machine was not required to generate this type of quantitative data and that this technology can be adapted for use in most molecular biology laboratories.

Key words: insulin-like growth factor I, messenger RNA, RT-PCR 

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Copyright information

© Springer-Verlag New York Inc. 2001

Authors and Affiliations

  • Anthony  Dyer
    • 1
  • Kathleen  Soole
    • 1
  • George  Matsumoto
    • 2
  1. 1.Co-operative Centre for Tissue Growth and Repair and The Flinders University of South Australia, School of Biological Science, Sturt Road Bedford Park, Australia 5042AU
  2. 2.Monterey Bay Aquarium Research Institute, Moss Landing, CA 95039-0628, U.S.A.US

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