Theoretical and functional aspects of measuring insulin-like growth factor-I mRNA expression in myeloid cells.
This article presents a detailed overview of the conceptual and technical considerations involved in the measurement of insulin-like growth factor-I (IGF-I) mRNAs in leukocytes. Two different quantitative techniques that take advantage of the in vitro synthesis of antisense and sense synthetic IGF-I RNA, respectively, are described: the ribonuclease protection assay (commonly referred to as solution hybridization) and competitive RT-PCR. We have improved the ribonuclease protection assay by constructing tandem, cassette riboprobes to generate multigene antisense RNAs of varying sizes. This approach permits the simultaneous quantitation of two or more mRNAs in a single RNA sample, one of which can serve as an internal standard for comparison of IGF-I transcripts among various treatments. The second approach of competitive RT-PCR represents an improvement in previous technologies by cloning a competing IGF-I sequence into an RNA expression vector. The resulting synthetic sense competitor IGF-I RNA (1.1 kb) serves as an internal standard during both the reverse transcription and amplification steps. We have used both the ribonuclease protection assay and competitive RT-PCR to define the macrophage as the major cellular source of leukocyte-derived IGF-I and to characterize these macrophage-derived mRNAs as being derived almost exclusively from exon 1. In addition, these techniques have allowed us to study the ontogeny of IGF-I expression in differentiating bone marrow macrophages and show that hematopoietic progenitors are induced to express abundant IGF-I transcripts as they differentiate into macrophages in the presence of CSF-1. These techniques can be readily adapted for measuring steady-state transcripts for a variety of leukocyte-derived hormones.
Department of Animal Sciences, 207 Plant and Animal Biotechnology Laboratory, Urbana, Illinois 61801.
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Arkins
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Kelley
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D015152:Blotting, Northern; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis / Q000235:genetics*; D007962:Leukocytes / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015347:RNA Probes; D012326:RNA Splicing; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D012260:Ribonucleases
D015347:RNA Probes; D016372:RNA, Antisense; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D012260:Ribonucleases