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.

Spinsanti, G., Zannolli, R., Panti, C., Ceccarelli, I., Marsili, L., Bachiocco, V., et al. (2008). Quantitative Real-Time PCR detection of TRPV1-4 gene expression in human leukocytes from healthy and hyposensitive subjects. MOLECULAR PAIN, 4, 51-51.

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

SPINSANTI, GIACOMO;ZANNOLLI, RAFFAELLA;PANTI, CRISTINA;CECCARELLI, ILARIA;MARSILI, LETIZIA;FRATI, FRANCESCO;ALOISI, ANNA MARIA
2008-01-01

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.
Spinsanti, G., Zannolli, R., Panti, C., Ceccarelli, I., Marsili, L., Bachiocco, V., et al. (2008). Quantitative Real-Time PCR detection of TRPV1-4 gene expression in human leukocytes from healthy and hyposensitive subjects. MOLECULAR PAIN, 4, 51-51.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/3203
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