Real-time RT-PCR quantification of insulin-like growth factor (IGF)-1, IGF-1 receptor, IGF-2, IGF-2 receptor, insulin receptor, growth hormone receptor, IGF-binding proteins 1, 2 and 3 in the bovine species

• Published in: Domestic animal endocrinology 2002-04, Vol.22 (2), p.91-102
• Main Authors: Pfaffl, M.W, Georgieva, T.Mircheva, Georgiev, I.Penchev, Ontsouka, E, Hageleit, M, Blum, J.W
• Format: Article
• Language: English
• Subjects: Animals; Calibration; Cattle; DNA, Recombinant; Humans; Ileum - chemistry; Insulin-Like Growth Factor Binding Protein 1 - genetics; Insulin-Like Growth Factor Binding Protein 2 - genetics; Insulin-Like Growth Factor Binding Protein 3 - genetics; Insulin-Like Growth Factor I - genetics; Insulin-Like Growth Factor II - genetics; Liver - chemistry; Mice; Receptor, IGF Type 1 - genetics; Receptor, IGF Type 2 - genetics; Receptor, Insulin - genetics; Receptors, Somatomedin - genetics; Receptors, Somatotropin - genetics; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - analysis; Sensitivity and Specificity; Sheep; Somatomedins - genetics
• ISSN: 0739-7240; 1879-0054
• DOI: 10.1016/S0739-7240(01)00128-X

Reverse transcription (RT) followed by polymerase chain reaction (PCR) is the technique of choice for analysing mRNA in extremely low abundance. Real-time RT-PCR using SYBR Green I detection combines the ease and necessary exactness to be able to produce reliable as well as rapid results. To obtain highly accurate and reliable results in a real-time RT-PCR a highly defined calibration curve is needed. We designed and developed nine different calibration curves, based on recombinant DNA plasmid standards and established them on a constant real-time PCR platform for the following factors: growth hormone receptor (GHR), insulin-like growth factor (IGF)-1, IGF-1 receptor (IGF-1R), IGF-2, IGF-2 receptor (IGF-2R), insulin receptor (INSR), and IGF-binding proteins (IGF-BP) 1, 2 and 3. Developed assays were applied in the LightCycler system on bovine ileum and liver total RNA and showed high specificity and sensitivity of quantification. All assays had a detection limit of under 35 recombinant DNA molecules present in the capillary. The SYBR Green I determination resulted in a reliable and accurate quantification with high test linearity (Pearson correlation coefficient r>0.99) over seven orders of magnitude from 10 8 recombinant DNA start molecules and an assay variation of maximal 5.3%. Applicability of the method was shown by analysing mRNA levels in newborn calves: mRNA concentrations per gram tissue of mRNAs of IGF-1, IGF-1R, IGF-2, IGF-2R, GHR, INSR, and IGF-BP1, 2 and 3 were all different between in liver and ileum and the traits all exhibited individual differences.