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Clinical and Diagnostic Laboratory Immunology, January 2002, p. 138-143, Vol. 9, No. 1
1071-412X/01/$04.00+0     DOI: 10.1128/CDLI.9.1.138-143.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Quantification of Substance P mRNA in Human Immune Cells by Real-Time Reverse Transcriptase PCR Assay

Jian-Ping Lai,1 Steven D. Douglas,1 Farida Shaheen,2 David E. Pleasure,3 and Wen-Zhe Ho1*

Division of Immunologic and Infectious Diseases,1 Center for AIDS Research, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104,2 Division of Neurology and Neurology Research, Department of Pediatrics, Joseph Stokes, Jr., Research Institute at the Children’s Hospital of Philadelphia3

Received 30 July 2001/ Returned for modification 24 September 2001/ Accepted 15 October 2001

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.

* Corresponding author. Mailing address: Division of Immunologic and Infectious Diseases, Children’s Hospital of Philadelphia, 34th & Civic Center Blvd., Philadelphia, PA 19104. Phone: (215) 590-4462. Fax: (215) 590-2025. E-mail: HO{at}EMAIL.CHOP.EDU.

Clinical and Diagnostic Laboratory Immunology, January 2002, p. 138-143, Vol. 9, No. 1
1071-412X/01/$04.00+0     DOI: 10.1128/CDLI.9.1.138-143.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Lai, J.-P., Douglas, S. D., Wang, Y.-J., Ho, W.-Z. (2005). Real-Time Reverse Transcription-PCR Quantitation of Substance P Receptor (NK-1R) mRNA. CVI 12: 537-541 [Abstract] [Full Text]  
  • Lai, J.-P., Yang, J.-H., Douglas, S. D., Wang, X., Riedel, E., Ho, W.-Z. (2003). Quantification of CCR5 mRNA in Human Lymphocytes and Macrophages by Real-Time Reverse Transcriptase PCR Assay. CVI 10: 1123-1128 [Abstract] [Full Text]  
  • Pintado, C. O., Pinto, F. M., Pennefather, J. N., Hidalgo, A., Baamonde, A., Sanchez, T., Candenas, M. L. (2003). A Role for Tachykinins in Female Mouse and Rat Reproductive Function. Biol. Reprod. 69: 940-946 [Abstract] [Full Text]  


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