Quantitative TaqMan PCR without a real-time thermal cycler: An assay for fish insulin-like growth factor I messenger RNA

被引:4
作者
Dyer, A
Soole, K
Matsumoto, G
机构
[1] Cooperat Ctr Tissue Growth & Repair, Bedford Pk, SA 5042, Australia
[2] Flinders Univ S Australia, Sch Biol Sci, Bedford Pk, SA 5042, Australia
[3] Monterey Bay Aquarium Res Inst, Moss Landing, CA 95039 USA
关键词
insulin-like growth factor I; messenger RNA; RT-PCR;
D O I
10.1007/s101260000029
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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.
引用
收藏
页码:16 / 21
页数:6
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