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

• Published in: Journal of immunological methods Vol. 230; no. 1; pp. 149 - 157
• Main Authors: Lai, Jian-Ping, Douglas, Steven D, Zhao, Ming, Ho, Wen-Zhe
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 19-11-1999; Elsevier
• Subjects: Adult; Alternative Splicing; Base Sequence; Biological and medical sciences; Cloning, Molecular; Diverse techniques; DNA Primers - genetics; DNA, Complementary - genetics; Evaluation Studies as Topic; Fundamental and applied biological sciences. Psychology; Gene Expression; Humans; Lymphocytes; Lymphocytes - chemistry; Molecular and cellular biology; Monocytes; Monocytes - chemistry; Neuroimmunology; Neuroimmunomodulation; Plasmids - genetics; PPT-A; Protein Precursors - genetics; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - statistics & numerical data; RNA, Messenger - analysis; RNA, Messenger - genetics; RT-PCR; Sensitivity and Specificity; Substance P; Substance P - genetics; Tachykinins - genetics; δ-PPT; Monocytes; PPT-A; Substance P; Lymphocytes; RT-PCR; Neuroimmunology; δ-PPT; Human; Polymerase chain reaction; Messenger RNA; Method; Gene expression; Neuropeptide; Tachykinin; Lymphocyte; Quantitative analysis; Phagocyte
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(99)00120-9

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 macrophages express substance P and neurokinin-1 receptor. Journal of Immunology, 159, p. 5654; Lai, J.P., Douglas, S.D., Ho, W.Z., 1998. Human lymphocytes 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. Identification of a delta isoform of preprotachykinin mRNA in human mononuclear phagocytes and lymphocytes. Journal of Neuroimmunology, 91, p. 121]. Using RT-PCR assay with several specific human SP 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 (α, β, γ, and δ) of the PPT-A gene. In an effort to quantitatively measure PPT-A mRNA levels, we have 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 four isoforms of PPT-A mRNA transcripts, with a sensitivity of 120 molecules per reaction. Thus the PPT-A mRNA transcripts in an unknown sample can be quantitatively analyzed using the mimic-based RT-PCR. The accuracy and reproducibility of this assay were confirmed by the plasmids containing α, β, γ and δ cDNA inserts and by in vitro synthesized mRNA from a plasmid containing β isoform cDNA insert. Our data indicate that the SP mimic-based RT-PCR assay has potential advantages in studies of SP levels in a variety of human cells as well as in clinical specimens.