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. 2008 Nov 4:4:51.
doi: 10.1186/1744-8069-4-51.

Quantitative Real-Time PCR detection of TRPV1-4 gene expression in human leukocytes from healthy and hyposensitive subjects

Giacomo Spinsanti  1 , Raffaella Zannolli, Cristina Panti, Ilaria Ceccarelli, Letizia Marsili, Valeria Bachiocco, Francesco Frati, Anna Maria Aloisi
Affiliations

Quantitative Real-Time PCR detection of TRPV1-4 gene expression in human leukocytes from healthy and hyposensitive subjects

Giacomo Spinsanti et al. Mol Pain. .

Abstract

Background: Besides functioning as chemosensors for a broad range of endogenous and synthetic ligands, transient receptor potential vanilloid (TRPV) 1-4 channels have also been related to capsaicin (TRPV1), pain, and thermal stimuli perception, and itching sensation (TRPV1-4). While the expression of the TRPV1-4 genes has been adequately proved in skin, sensory fibres and keratinocytes, less is known about TRPV3 and TRPV4 expression in human blood cells.

Results: To study the gene expression of TRPV1-4 genes in human leukocytes, a quantitative Real-Time PCR (qRT-PCR) method, based on the calculation of their relative expression, has been developed and validated. The four commonly used house-keeping genes (HKGs), beta-Actin (Act-B), glyceraldehyde-3P-dehydrogenase (GAPDH), hypoxanthine ribosyltransferase (HPRT1), and cyclophilin B (hCyPB), were tested for the stability of their expression in several human leukocyte samples, and used in the normalization procedure to determine the mRNA levels of the TRPV 1-4 genes in 30 healthy subjects. cDNAs belonging to all the TRPV1-4 genes were detected in leukocytes but the genes appear to be expressed at different levels. Our analysis did not show significant sex differences in TRPV1-4 cDNA levels in the 30 healthy subjects. The same qRT-PCR assay was used to compare TRPV1-4 expression between healthy controls and patients hyposensitive to capsaicin, pain and thermal stimuli: an almost doubled up-regulation of the TRPV1 gene was found in the pathological subjects.

Conclusion: The qRT-PCR assay developed and tested in this study allowed us to determine the relative expression of TRPV1-4 genes in human leukocytes: TRPV3 is the least expressed gene of this pool, followed by TRPV4, TRPV1 and TRPV2. The comparison of TRPV1-4 gene expression between two groups of healthy and hyposensitive subjects highlighted the evident up-regulation of TRPV1, which was almost doubly expressed (1.9x normalized fold induction) in the latter group. All the four house-keeping genes tested in this work (Act-B, GAPDH, hCyPB, HPRT1) were classified as optimal controls and showed a constant expression in human leukocytes samples. We recommend the use of these genes in similar qRT-PCR studies on human blood cells.

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Figures

Figure 1
Figure 1
Relative expression of the TRPV1–4 genes in human leukocytes from healthy subjects (n = 30). Relative expression measurements of the TRPV1–4 genes using qRT-PCR calculated using REST2005 (relative expression software tool). Fold change (y axis) represents the relative expression of the TRPV1, TRPV2 and TRPV4 genes in comparison to TRPV3 (least expressed gene of this pool and therefore considered equal to 1), normalized by Act-B, GAPDH, hCyPB and HPRT1 reference gene expressions. Expression standard errors are expressed within brackets at the top of each bar. * means p < 0.001.
Figure 2
Figure 2
Reciprocal expression levels of TRPV1–4 genes in human leukocytes from healthy donors (n = 30). Expression rates of TRPV1–4 genes calculated using qRT-PCR with relative quantification analysis using REST (relative expression software tool). Fold changes represents the reciprocal relative expression of the TRPV1–4 mRNA in physiological conditions in the 30 healthy donors. Values below (above) the diagonal line of each cell refer to the expression levels of genes on the y-axis (x-axis) compared to those of the corresponding genes on the x-axis (y-axis); S.E. means Expression Standard Error as calculated by the software REST; all p values are below 0.001.
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