Quantification of substance P mRNA in human immune cells by real-time reverse transcriptase PCR assay

Jian Ping Lai, Steven D. Douglas, Farida Shaheen, David E Pleasure, Wen Zhe Ho

Research output: Contribution to journalArticle

35 Scopus citations

Abstract

We have applied a newly developed real-time reverse transcriptase (RT) PCR (RT-PCR) assay for quantification of substance P (SP) mRNA expression (the SP real-time RT-PCR assay) in human blood monocyte-derived macrophages, peripheral blood lymphocytes, and microglia isolated from fetal brain. The SP real-time RT-PCR assay had a sensitivity of 60 mRNA copies, with a dynamic range of detection between 60 and 600,000 copies of the SP gene transcript per reaction mixture. The coefficient of variation of the threshold cycle number between the SP real-time RT-PCR assays was less than 1.16%. This assay with an SP-specific primer pair efficiently recognizes all four isoforms of preprotachykinin A (the SP precursor) gene transcripts. In order to use this assay to measure the levels of SP mRNA in the human immune cells quantitatively, we designed a specific probe (molecular beacon) derived from exon 3 of the SP gene. We demonstrated that the real-time RT-PCR quantitatively detected SP mRNA in the human immune cells, among which the microglia isolated from fetal brain had the highest levels of SP mRNA. The SP real-time PCR assay yielded reproducible data, as the intra-assay variation was less than 1%. Thus, it is feasible to apply the real-time RT-PCR assay for quantification of SP mRNA levels in human immune cells, as well as in other nonneuronal cells. Since SP is a major modulator of neuroimmunoregulation, this assay has the potential for widespread application for basic and clinical investigations.

Original languageEnglish (US)
Pages (from-to)138-143
Number of pages6
JournalClinical and Diagnostic Laboratory Immunology
Volume9
Issue number1
DOIs
StatePublished - 2002
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology (medical)
  • Immunology and Allergy
  • Clinical Biochemistry
  • Immunology

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