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Quantitative Real Time Polymerase Chain Reaction for measurement of human Interleukin – 5 receptor alpha spliced isoforms mRNA

DOI: 10.1186/1472-6750-3-17

Keywords: Soluble interleukin 5 receptor alpha, membrane-anchored interleukin 5 receptor alpha, real-time PCR, SYBR Green I, eosinophilic chronic rhinosinusitis, peripheral blood, alternative splicing

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Abstract:

For the quantitative real-time PCR assay, two standard curves specific for each splice variant were constructed. PCR amplifications were performed on CDNA from peripheral blood, eosinophilic chronic rhinosinusitis and normal nasal tissue using a common forward and two specific reverse primers, in combination with SYBR Green I as the detection format.We have developed an accurate and reliable assay for quantification of interleukin-5 receptor alpha mRNA isoforms over a broad dynamic range of input molecules. Importantly, excess of one isoform did not influence accurate quantification of the other isoform. Quantification of hIL-5Rα variants in human samples demonstrated an overexpression of both membrane-anchored and soluble encoding variants in eosinophilic chronic rhinosinusitis tissue and peripheral blood in patients with eosinophilic chronic rhinosinusitis compared to healthy subjects. The implementation of this assay will allow a better understanding of the regulatory mechanisms of the hIL-5Rα gene and hence its role in the pathogenesis of chronic inflammatory diseases.Immune responses are mediated by a large group of peptides known as cytokines. These molecules play an important role in promoting cell growth, differentiation, activation and regulation of human inflammatory responses. Human interleukin-5 (hIL-5), a haemopoietin that belongs to the alpha-helical group of cytokines, plays an essential role in the induction and maintenance of eosinophilic airway infiltration [1-3]. It has been shown that this cytokine is linked to the occurrence of chronic inflammatory diseases such as asthma and eosinophilic chronic rhinosinusitis [4-6]. The action of the hIL-5 is mediated by interaction with its receptor, the human IL-5 receptor. This receptor belongs to the class I cytokine receptor family together with receptors for IL-3 and GM-CSF [7,8] and consists of a heterodimer containing a unique α-subunit required for ligand-specific binding [9], and a β-subunit involved

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