Quantification of common carp (Cyprinus carpio) IGF-I and IGF-II mRNA by real-time PCR:: differential regulation of expression by GH

IGFs are potent mitogens for many different cell types and play important roles in growth and development. A multitude of regulatory factors modulate the expression of IGFs. In some teleosts, liver IGF-I expression has been demonstrated to depend on the presence of GH. However, the GH dependence of IGF-II expression in teleosts is controversial. Moreover, most IGF expression studies in bony fish have been focused on the liver, and information on extrahepatic tissues are conflicting and inconsistent. This is partly due to the fact that the traditional methods of mRNA measurement such as Northern blot and RT-PCR are not sensitive enough to detect changes in IGF levels in extrahepatic tissues because of the low levels of IGFs in these tissues. In addition, there have been few studies on the IGF system of non-salmonid teleosts. Our laboratory has thus begun such studies on a local tropical fast-growing fish, the common carp (Cyprinus carpio). In this study, real-time quantitative PCR assays were developed for the accurate measurement of IGF-I and IGF-II mRNA levels in common carp tissues. This quantitative method was based on the measurement of a fluorescent labeled probe, which was cleaved by Taq polymerase during PCR by the 5'-->3' nuclease activity. The signal generated was directly proportional to the starting copy number of the target molecules in the sample. Hence, it was possible to detect and quantify the mRNA levels of both IGF-I and IGF-II reliably in very small amounts of tissues obtained from juvenile common carp. Using these assays, the expression pattern of IGF-I and IGF-II in various common carp tissues was Studied, and their differential response to GH stimulation was also investigated.