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 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 (alpha, beta, gamma, and delta) 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 alpha, beta, gamma and delta cDNA inserts and by in vitro synthesized mRNA from a plasmid containing beta 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.
Lai
J P
JP
Department of Pediatrics, University of Pennsylvania Medical School, Philadelphia, PA 19104, USA.
Douglas
S D
SD
Zhao
M
M
Ho
W Z
WZ
D000328:Adult; D017398:Alternative Splicing; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005069:Evaluation Studies as Topic; D015870:Gene Expression; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry; D009000:Monocytes / Q000737:chemistry; D015213:Neuroimmunomodulation; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D013373:Substance P / Q000235:genetics*; D015320:Tachykinins / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D011498:Protein Precursors; D012333:RNA, Messenger; D015320:Tachykinins; C044206:preprotachykinin; D013373:Substance P