Quantification of insulin-like growth factor-1 (IGF-1) mRNA: development and validation of an internally standardised competitive reverse transcription-polymerase chain reaction.

Abstract

To investigate the role of local IGF-1 mRNA expression in various tissues, we developed and validated a method which allows for a specific, sensitive and reliable quantification of IGF-1 mRNA: an internally standardised Reverse Transcription-Polymerase Chain Reaction (RT-PCR). A synthetic competitive template IGF-I standard cRNA (IGF-1 cRNA) was designed, which contains the same flanking primer sequences used to amplify the wild type IGF-1 mRNA, but differs by 56 bp in length. To obtain the IGF-1 mRNA concentration present in tissue RNA samples, series of 250 ng total-RNA were spiked with three known quantities of the standard IGF-1 cRNA, incubated for competitive RT-PCR reactions and the two amplificates obtained (184 bp from IGF-1 cRNA and 240 bp from the wild type IGF-I mRNA) were subsequently separated and quantified by HPLC-UV. For every individual tissue RNA sample, the ratio R (R = competitor PCR product / wild type PCR product) was plotted against the number of starting molecules of the competitor IGF-1 cRNA. The initial amount of IGF-1 mRNA present in the sample can then be read off where R = 1. The validated assay had a detection limit of 1600 IGF-1 cRNA molecules/reaction, the intra-assay variation was 7.4% (n = 5) and linearity (r = 0.997) was given between 140 ng to 840 ng total-RNA input. The present method was first applied to study the effect of long term castration on the IGF-1 expression rates in bovine tissues. The hepatic IGF-1 mRNA concentrations were well correlated (r = 0.81) with the plasma concentrations as quantified by RIA and were higher in intact than in castrated animals. In two skeletal muscles (m. splenius and m. gastrocnemius) IGF-1 mRNA concentrations were 20- and 35- times lower than in liver, respectively, without any differences between steers and bulls. In bulls, the IGF-1 mRNA expression was higher in m. splenius (p < 0.01) than m. gastrocnemius, indicating that locally produced IGF-1 might be important for sexually dimorphic muscle growth patterns.