Quantification of substance p mRNA in human immune cells by real-time reverse transcriptase PCR assay.
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.
Lai
Jian-Ping
JP
Division of Immunologic and Infectious Diseases, Children's Hospital of Philadelphia, 34th and Civic Center Blvd., Philadelphia, PA 19104, USA.
Douglas
Steven D
SD
Shaheen
Farida
F
Pleasure
David E
DE
Ho
Wen-Zhe
WZ
D000328:Adult; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D017628:Microglia / Q000378:metabolism; D008875:Middle Aged; D011498:Protein Precursors / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013373:Substance P / Q000235:genetics*; D015320:Tachykinins / Q000235:genetics
D011498:Protein Precursors; D012333:RNA, Messenger; D015320:Tachykinins; C044206:preprotachykinin; D013373:Substance P