TY - JOUR
T1 - Quantitative TaqMan PCR without a real-time thermal cycler
T2 - An assay for fish insulin-like growth factor I messenger RNA
AU - Dyer, Anthony
AU - Soole, Kathleen
AU - Matsumoto, George
PY - 2001/1
Y1 - 2001/1
N2 - 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.
AB - 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.
KW - Insulin-like growth factor I
KW - Messenger RNA
KW - RT-PCR
UR - http://www.scopus.com/inward/record.url?scp=0034830928&partnerID=8YFLogxK
U2 - 10.1007/s101260000029
DO - 10.1007/s101260000029
M3 - Article
AN - SCOPUS:0034830928
VL - 3
SP - 16
EP - 21
JO - Marine Biotechnology
JF - Marine Biotechnology
SN - 1436-2228
IS - 1
ER -