Establishment of a real-time RT-PCR for the determination of absolute amounts of IGF-I and IGF-II gene expression in liver and extrahepatic sites of the tilapia.

We developed a one-tube two-temperature real-time RT-PCR that allows to absolutely quantify the gene expression of hormones using the standard curve method. As our research focuses on the expression of the insulin-like growth factors (IGFs) in bony fish, we established the technique for IGF-I and IGF-II using the tilapia (Oreochromis niloticus) as model species. As approach, we used primer extension adding a T7 phage polymerase promoter (21 nt) to the 5' end of the antisense primers. This procedure avoids the disadvantages arising from plasmids. Total RNA extracted from liver was subjected to conventional RT-PCR to create templates for in vitro transcription of IGF-I and IGF-II cRNA. Correct template sizes including the T7 promoter were verified (IGF-I: 91 nt; IGF-II: 94 nt). The PCR products were used to create IGF-I and IGF-II cRNAs which were quantified in dot blot by comparison with defined amounts of standardised kanamycin mRNA. Standardised threshold cycle (Ct) values for IGF-I and IGF-II mRNA were achieved by real-time RT-PCR and used to create standard curves. To allow sample normalisation the standard curve was also established for beta-actin as internal calibrator (template: 86 nt), and validation experiments were performed demonstrating similar amplification efficiencies for target and reference genes. Based on the standard curves, the absolute amounts of IGF-I and IGF-II mRNA were determined for liver (IGF-I: 8.90+/-1.90 pg/microg total RNA, IGF-II: 3.59+/-0.98 pg/microg total RNA) and extrahepatic sites, such as heart, kidney, intestine, spleen, gills, gonad, and brain considering the different lengths of cRNAs and mRNAs by correction factors. The reliability of the method was confirmed in additional experiments. The amplification of descending dilutions of cRNA and total liver RNA resulted in parallel slopes of the amplification curves. Furthermore, amplification plots of the standard cRNA and the IGF-I and IGF-II mRNAs showed signals starting at the expected Ct values. Thus, the one-tube RT-PCR described here is highly sensitive (detection level approximately 2 pg/microg total RNA) and allows precise absolute quantification. The method is rapid as there are neither separate reverse transcriptions nor post-amplification steps, and can be executed with low risk of contamination. Therefore, it will be helpful when investigating gene expression in any species and tissue whenever absolute levels are of concern.