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.
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(2) to >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.
Pfaffl
M W
MW
Institute of Physiology, Center of Life and Food Sciences, Technical University of Munich, 85354 Freising-Weihenstephan, Germany. pfaffl@wzw.tum.de
Georgieva
T Mircheva
TM
Georgiev
I Penchev
IP
Ontsouka
E
E
Hageleit
M
M
Blum
J W
JW
D000818:Animals; D002138:Calibration; D002417:Cattle; D004274:DNA, Recombinant; D006801:Humans; D007082:Ileum / Q000737:chemistry; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000235:genetics; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000235:genetics; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000235:genetics; D007335:Insulin-Like Growth Factor II / Q000235:genetics; D008099:Liver / Q000737:chemistry; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis; D017526:Receptor, IGF Type 1 / Q000235:genetics; D017527:Receptor, IGF Type 2 / Q000235:genetics; D011972:Receptor, Insulin / Q000235:genetics*; D017451:Receptors, Somatomedin / Q000235:genetics*; D011986:Receptors, Somatotropin / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012756:Sheep; D013002:Somatomedins / Q000235:genetics*
D004274:DNA, Recombinant; D018970:Insulin-Like Growth Factor Binding Protein 1; D018971:Insulin-Like Growth Factor Binding Protein 2; D018972:Insulin-Like Growth Factor Binding Protein 3; D012333:RNA, Messenger; D017527:Receptor, IGF Type 2; D017451:Receptors, Somatomedin; D011986:Receptors, Somatotropin; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin