Quantification of substance P mRNA in human mononuclear phagocytes and lymphocytes using a mimic-based RT-PCR

We have recently demonstrated that human monocytes and lymphocytes express the substance P (SP) gene at both the mRNA and protein level [Ho, W.Z,, Lai , J.P., Zhu, X.H., Uvaydova, M., Douglas S.D., 1997. Human monocytes and ma crophages express substance P and neurokinin-l receptor. Journal of Immunol ogy, 159, p. 5654; Lai, J.P., Douglas, S.D., Ho, W.Z., 1998. Human lymphocy tes express substance P and its receptor. Journal of Neuroimmunology, 86, p . 80; Lai, J.-P., Douglas, S.D., Rappaport, E,, Wu, J., Ho, W.-Z., 1998. Id entification of a delta isoform of preprolachykinin mRNA in human mononucle ar phagocytes and lymphocytes. Journal of Neuroimmunology, 91, p. 121]. Usi ng RT-PCR assay with several specific human P primer pairs, we were able to differentiate four isoforms of preprotachykinin (PPT-A, the SP precursor) mRNA transcripts on ethidium bromide-stained agarose gels and clone the PCR amplified cDNA of the four isoforms (alpha, beta, gamma, and delta) of the PPT-A gene. In an effort to quantitatively measure PPT-A mRNA levels, we h ave developed a mimic-based RT-PCR assay to analyze total PPT-A mRNA levels in human monocytes and lymphocytes. We designed a specific human SP primer pair (HSP4/HSP3) to amplify a single fragment of cDNA derived from all fou r isoforms of PPT-A mRNA transcripts, with a sensitivity of 120 molecules p er reaction. Thus the PPT-A mRNA transcripts in an unknown sample can be qu antitatively analyzed using the mimic-based RT-PCR. The accuracy and reprod ucibility of this assay were confirmed by the plasmids containing alpha, be ta, gamma and delta cDNA inserts and by in vitro synthesized mRNA from a pl asmid containing beta isoform cDNA insert. Our data indicate that the SP mi mic-based RT-PCR assay has potential advantages in studies of SP levels in a variety of human cells as well as in clinical specimens. (C) 1999 Elsevie r Science B.V. All rights reserved.