Molecular markers of eosinophilopoiesis: multiplex Q-PCR analysis of GATA-1, MBP and IL-5 receptor mRNA expression in peripheral blood

Ellis, A.K., Crawford, L. and Denburg, J.A. (2007) Molecular markers of eosinophilopoiesis: multiplex Q-PCR analysis of GATA-1, MBP and IL-5 receptor mRNA expression in peripheral blood. Journal of Allergy and Clinical Immunology, 119(1), S218. (doi: 10.1016/j.jaci.2006.12.225)

Abstract

<b>RATIONALE:</b> Using colony assays and flow cytometry, we have shown that eosinophil/basophil (Eo/B) progenitor phenotype and function are associated with atopic risk at birth and early childhood clinical outcomes. We have also recently demonstrated that real-time polymerase chain reaction (Q-PCR) can reveal kinetic patterns of expression in cord blood (CB) of several Eo/B lineage-specific genes, specifically GATA-1, MBP and IL-5Rα, as surrogate molecular markers of Eo/B differentiation. These same methods have yet to be established in peripheral blood (PB) samples. Our objective was to determine the kinetic patterns of expression of CB Eo/B-lineage specific genes in PB.<p></p> <b>METHODS:</b> PB non-adherent mononuclear cells (PB NAMNC) were isolated from random fresh samples, and incubated in the presence of IL-5. At 24, 48, and 72h post-stimulation, RNA was isolated, reverse transcribed, and expression of IL-5Rα, GATA-1, and MBP was determined utilizing multiplex Q-PCR. Relative expression ratios of stimulated to un-stimulated cells were calculated using the delta-delta Ct method.<p></p> <b>RESULTS:</b> Stimulation of PB NANMC with IL-5 resulted in an up-regulation of GATA-1 expression, peaking at 24h, with a slower return to baseline expression than that observed in CB. MBP expression was minimally altered at all time points, compared to CB, where slow up-regulation, maximal at 72h, had been observed. There was completely stable expression IL-5Rα, similar to that seen in CB.<p></p> <b>CONCLUSIONS:</b> Multiplex Q-PCR analysis of mRNA from PB demonstrates expression of critical Eo/B lineage-specific events. Further investigation of the validity and utility of Q-PCR analyses of PB for surrogate, molecular markers Eo/B differentiation is underway.<p></p>

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Stewart, Ms Lynn
• Authors: Ellis, A.K., Crawford, L., and Denburg, J.A.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Journal Name: Journal of Allergy and Clinical Immunology
• Journal Abbr.: J. Allergy Clin. Immunol.
• Publisher: Elsevier
• ISSN: 0091-6749
• ISSN (Online): 1097-6825