Real-time reverse transcription-PCR quantitation of substance P receptor (NK-1R) mRNA - PubMed (original) (raw)

Comparative Study

Real-time reverse transcription-PCR quantitation of substance P receptor (NK-1R) mRNA

Jian-Ping Lai et al. Clin Diagn Lab Immunol. 2005 Apr.

Abstract

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.

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Figures

FIG. 1.

FIG. 1.

Sensitivity and linearity analysis of the NK-1R real-time RT-PCR. A reading of change in fluorescence (Rn) as a function of cycle numbers is demonstrated for a range of known input copy numbers of the NK-1R RNA transcript derived from the plasmid containing NK-1R cDNA fragment. Tenfold serial dilutions of the NK-1R RNA starting from 102 to 107 molecules per reaction were amplified by the real-time RT-PCR. (A) The standard curve of the serial dilutions of the NK-1R RNA with a correlation coefficient (_R_2) of 0.9988. (B) Amplification plot of the serial dilutions of NK-1R RNA. The dynamic detection range is 5 orders of magnitude from 102 to 107 molecules, and the detection sensitivity is 100 NK-1R mRNA copies per reaction. NC, negative control which lacked PCR-amplified product when reverse transcriptase was omitted from the RT reaction using 107 molecules of NK-1R RNA standard.

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