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


CONCLUSIONS
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.
D017398:Alternative Splicing; D052160:Benzothiazoles; D003959:Diamines; D004802:Eosinophilia / Q000276:immunology; D005456:Fluorescent Dyes; D006801:Humans; D053666:Interleukin-5 Receptor alpha Subunit; D008969:Molecular Sequence Data; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011804:Quinolines; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D018123:Receptors, Interleukin / Q000235:genetics* / Q000378:metabolism; D053649:Receptors, Interleukin-5; D015203:Reproducibility of Results; D012852:Sinusitis / Q000276:immunology; D013997:Time Factors
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; C485918:IL5RA protein, human; D053666:Interleukin-5 Receptor alpha Subunit; D009930:Organic Chemicals; D020033:Protein Isoforms; D011804:Quinolines; D012333:RNA, Messenger; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; C098022:SYBR Green I