Quantitative measurement of human cytokine gene expression by polymerase chain reaction

Peter C. Melby, Barbara J. Darnell, Victor V. Tryon

Research output: Contribution to journalArticle

52 Scopus citations

Abstract

We have developed a method in which human cytokine gene expression can be quantitated using gene amplification technology. Total RNA was extracted from a human T cell line, reverse transcribed to cDNA, and amplified using the polymerase chain reaction (PCR). Inclusion of a radiolabeled nucleotide in the PCR reaction mixture followed by electrophoresis and quantitative imaging of the amplification product with the BetaScope imager and software enabled quantitation of the input cDNA. Linear standard curves within the exponential phase of DNA amplification using purified cytokine cDNA templates were generated over a several log concentration range of input DNA. A 10-67-fold increase in interleukin-2 (IL-2), IL-3, IL-4, IL-10, and interferon-γ gene expression following cell activation could be identified by interpolation from the standard curve. The lower limit of linearity on the standard curve was as little as 0.01 fg of input DNA which corresponded to approximately 20 cells. This very sensitive methodology is a valuable tool in the detection and quantitation of cytokine gene expression when only small amounts of tissue or cells are available.

Original languageEnglish (US)
Pages (from-to)235-244
Number of pages10
JournalJournal of Immunological Methods
Volume159
Issue number1-2
DOIs
StatePublished - Feb 26 1993
Externally publishedYes

Keywords

  • Cytokine
  • Gene expression
  • Polymerase chain reaction
  • RNA
  • T lymphocyte

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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