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Clinical and Diagnostic Laboratory Immunology, April 2005, p. 537-541, Vol. 12, No. 4
1071-412X/05/$08.00+0     doi:10.1128/CDLI.12.4.537-541.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Real-Time Reverse Transcription-PCR Quantitation of Substance P Receptor (NK-1R) mRNA

Jian-Ping Lai, Steven D. Douglas, Yan-Jian Wang, and Wen-Zhe Ho*

Division of Allergy and Immunology, Joseph Stokes Jr. Research Institute at the Children's Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104

Received 9 August 2004/ Returned for modification 21 December 2004/ Accepted 31 January 2005

The substance P (SP)-preferring receptor, neurokinin-1 receptor (NK-1R), has an important role in inflammation, immune regulation, and viral infection. We applied a newly developed real-time reverse transcription (RT)-PCR assay to quantify NK-1R mRNA in human neuronal cell line (NT-2N), a human B-cell line (IM9), monocyte-derived macrophages (MDM), peripheral blood lymphocytes (PBL), and human astroglioma cells (U87 MG). The NK-1R real-time RT-PCR assay has a sensitivity of 100 mRNA copies, with a dynamic range of detection between 102 and 107 copies of NK-1R gene transcripts per reaction. This assay is highly reproducible, with an intraassay coefficient variation of threshold cycle (Ct) of less than 1.9%. The NK-1R real-time RT-PCR is highly sensitive for quantitative determination of NK-1R mRNA in human immune cells (MDM and PBL) that express low levels of NK-1R mRNA. In addition, the assay has the ability to accurately quantitate the dynamic changes in NK-1R mRNA expression in interleukin-1ß-stimulated U87 MG. These data indicate that the NK-1R real-time RT-PCR has potential for a wide application in investigation of NK-1R expression at the mRNA level under physiological and pathological conditions in both the central nervous system and the immune system.

* Corresponding author. Mailing address: Division of Allergy and Immunology, The 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, April 2005, p. 537-541, Vol. 12, No. 4
1071-412X/05/$08.00+0     doi:10.1128/CDLI.12.4.537-541.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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