In this study a solution hybridization assay was evaluated for its application to the measurement of levels of specific mRNAs. The evaluation included parameters such as incubation time, hybridization stringency and probe concentration/structure. Both short (50 bases derived from synthetic oligonucleotides) and long (125–147 bases) RNA probes, derived from cloned sequences, could be used to obtain quantitative information on specific mRNA species.
The solution hybridization assay was used to compare the levels of insulin-like growth factor-I (IGF-I) and IGF-II mRNAs in various rat and human tissues. In the rat the liver was the main source of IGF-I mRNA (approximately 400 molecules/cell), but significant levels were also found in extrahepatic tissues such as fat and muscle (3–50 molecules/cell). Human liver contained approximately 100-fold less IGF-I mRNA than rat liver. Human fat, muscle and placenta contained levels of IGF-I mRNA (2–8 molecules/cell) similar to those in the liver. Levels of IGF-II mRNA in rat and human tissues were similar, in that the expression was greatest in the placenta (approximately 200 molecules/cell). Species differences were evident, however, since human liver and fat contained significant amounts of IGF-II mRNA (15–20 molecules/cell), while the rat counterparts had almost undetectable levels. Young and old rats were used to examine the influence of age on the expression of IGF-I and GH receptor mRNAs in the liver. Levels of both IGF-I mRNA and GH receptor mRNA were found to decrease with age (2.8-fold and 1.7-fold respectively).
It is concluded that low levels of IGF mRNAs can be detected using the solution hybridization assay and that there are considerable species differences within and between tissues with regard to steady-state levels of IGF-I and IGF-II mRNAs.
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