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Abstract
Objectives. (1) To investigate the effects of globin mRNA depletion in detecting differential gene expression in peripheral blood and (2) to investigate changes in peripheral blood gene expression in atopic asthmatic individuals undergoing allergen inhalation challenge. Methods. Asthmatic subjects (20–60 years of age, with stable, mild allergic asthma, n = 9) underwent allergen inhalation challenges. All had an early asthmatic response of ≥20% fall in forced expiratory volume in 1 second. Blood was collected immediately prior to and 2 hours after allergen challenge using PAXgene tubes (n = 4) and EDTA tubes (n = 5). Aliquots of the PAXgene blood samples were subjected to globin reduction (PAX-GR). Transcriptome analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST arrays. Data were preprocessed using factor analysis for robust microarray summarization and analyzed using linear models for microarrays. Pathway analyses were performed using Ingenuity Pathway Analysis. Results. Globin reduction uncovered probe sets of lower abundance. However, it significantly reduced the ability to detect differentially expressed genes (DEGs) when compared to non-globin-reduced PAXgene samples (PAX-NGR). Combined transcriptional analysis of four PAX-NGR and five EDTA sample pairs identified 1595 DEGs associated with allergen inhalation challenge (false discovery rate ≤ 5%), with the top-ranked network of perturbed biological functions consisting of inflammatory response, cellular movement, and immune cell trafficking. Conclusions. While we have demonstrated a diminished ability to detect DEGs after globin reduction, we have nevertheless identified significant changes in the peripheral blood transcriptome of people with mild asthma 2 hours after allergen inhalation challenge.
Acknowledgements
We thank the nine research participants for taking part in these studies, as well as Linda Hui, Nathalie Y, Freda Tom, Megan O’Connor, Rick Watson, Heather Campbell, and Karen Howie for their expertise and assistance with subject recruitment, allergen challenge, and sample collection, as part of the AllerGen Clinical Investigator Collaborative. We also thank the staff at CTAG (BC Cancer Agency, Vancouver, BC, Canada) for their help with the microarray experiments. This research was supported by funding from AllerGen NCE Inc. (Allergy, Genes and Environment Network) and the Canadian Institutes of Health Research. SHYK is the recipient of an AllerGen Canadian Allergy and Immune Diseases Training Award (CAIDATI).
Gail M. Gauvreau, Paul M. O’Byrne, J. Mark Fitzgerald, and Scott J. Tebbutt participated in research design and provision of samples. Sarah H.Y. Kam, Jian Ruan, Gail M. Gauvreau, and Scott J. Tebbutt participated in the performance of the research. Sarah H.Y. Kam, Amrit Singh, Jian-Qing He, Gail M. Gauvreau, and Scott J. Tebbutt participated in data analysis and in the writing of the paper.
Declaration of Interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.