Real-time reverse transcription-PCR quantitation of substance P receptor (NK-1R) mRNA.
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 10(2) and 10(7) 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-1beta-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.
Lai
Jian-Ping
JP
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, USA.
Douglas
Steven D
SD
Wang
Yan-Jian
YJ
Ho
Wen-Zhe
WZ
D002478:Cells, Cultured; D005786:Gene Expression Regulation; D006801:Humans; D016014:Linear Models; D012333:RNA, Messenger / Q000032:analysis; D018040:Receptors, Neurokinin-1 / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D012333:RNA, Messenger; D018040:Receptors, Neurokinin-1