Pii: 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 ∗Corresponding author. Tel.: +49-8161-71-3511; fax: +49-8161-71-4204. E-mail address: [email protected] (M.W. Pfaffl). 1Present address: Department of Biochemistry, Veterinary Faculty, Thracian University, 6000 Stara Zagora, Bulgaria. 2Present address: Department of Veterinary Physiology and Endocrinology, Veterinary Faculty, Thracian University, 6000 Stara Zagora, Bulgaria. 0739-7240/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S0739-7240(01)00128-X 92 M.W. Pfaffl et al. / Domestic Animal Endocrinology 22 (2002) 91–102 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 <102 to >108 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. © 2002 Elsevier Science Inc. All rights reserved.