Quantitative real time polymerase chain reaction for measurement of human interleukin-5 receptor alpha spliced isoforms mRNA

Abstract

Background: Expression of human Interleukin-5 receptor alpha (hIL-5Ralpha) is controlled by alternative splicing, which generates two different transcripts encoding a membrane-anchored and a soluble form of the receptor, respectively. Although the study of the expression and regulation of hIL-5Ralpha is of crucial importance in the field of immunological processing, methods and techniques until now described lack sufficient sensitivity for detection of small differences in the expression of these isoforms. The aim of this study was to develop a reliable and sensitive real-time quantitative PCR assay to analyse the expression level of each isoform.

Methods: 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.

Results and conclusion: 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-5Ralpha 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-5Ralpha gene and hence its role in the pathogenesis of chronic inflammatory diseases.

Figure 1

Alternative splicing of the human Interleukin 5 receptor alpha gene (hIL-5Rα) generating two transcripts encoding the soluble and membrane-anchored hIL-5Rα isoforms. Skipping of exons 12, 13 and 14 generate the soluble encoding form, whereas for the membrane-anchored encoding variant, only exon 11 is skipped. Arrows indicate the region amplified by the primer pairs 3 or 4.

Figure 2

PCR amplification plots of each hIL-5Rα splice variant in the presence of excess of the alternative splice form. PCR amplification plots of the membrane-anchored (A) and soluble (B) encoding splice variant in presence of the alternative splice variant (data generated on iCycler iQ Real-Time PCR Detection System, BioRad Laboratories, USA). Curves in blue indicate the standards diluted in water; curves in red represent standards diluted in 5 × 10⁵ molecules of the alternative splice form. The table indicates the quantities for both standard curves and the coefficient of variation (C.V) between the quantities (molecule number) obtained for each dilution point.

Figure 3

Relative number of molecules of the transcripts encoding the soluble and membrane anchored of hIL-5Rα after normalization to beta-actin (ACTB) gene in eosinophilic chronic rhinosinusitis and normal nasal mucosa (A) and in peripheral blood from eosinophilic chronic rhinosinusitis patients and healthy subjects (B). **: p < 0.01; *: p < 0.05; N.S.: non significant differences, CRS: eosinophilic chronic rhinosinusitis patients; Ctrol.: healthy subjects.