Competitive reverse transcriptase-polymerase chain reaction using a synthetic internal RNA standard to quantitate transcripts for leukocyte-derived hormones.

Q. Liu, S. Arkins, A. Biragyn, Christian T Minshall, P. Parnet, R. Dantzer, K. W. Kelley

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

Leukocytes synthesize a variety of hormones that were once thought to be unique products of endocrine tissues. Understanding the regulation of leukocyte-derived hormone synthesis requires an accurate means for measuring steady-state expression of specific mRNA transcripts. Here we describe a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) technique to accurately quantitate macrophage-derived insulin-like growth factor-I (IGF-I) mRNA, and demonstrate the utility of this approach for measuring expression of leukocyte-derived hormone transcripts. A riboprobe was constructed to generate approximately 1 kb of synthetic competitor IGF-I RNA (exons 1 and 3-6) that differed from cellular IGF-I RNA by insertion of 122 bp of beta-actin RNA. One set of oligonucleotide primers could thus be used to simultaneously reverse transcribe and amplify both 144 bp of cellular (exons 3 and 4) and 266 bp of competitor IGF-I RNA. Densitometric scanning of the PAGE-separated PCR products revealed that the ratio of competitor to cellular amplified DNA bore a linear relationship (r2 > or = 0.98) to the amount of competitor RNA for both rat liver and splenocytes. However, rat liver contained 104 x 10(6) IGF-I molecules per microgram of total cellular RNA compared to only 2 x 10(6) IGF-I molecules for splenocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Original languageEnglish (US)
Pages (from-to)33-41
Number of pages9
JournalNeuroimmunomodulation
Volume1
Issue number1
DOIs
StatePublished - Jan 1994

ASJC Scopus subject areas

  • Immunology
  • Endocrinology
  • Neurology
  • Endocrine and Autonomic Systems

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