Microbiome and Inflammatory Interactions in Obese and Severe Asthmatic Adults

In order to better understand the systemic immunological responses in a clinical cohort of obese and non-obese asthmatics and healthy subjects, we sought to analyze gene expression from whole blood. We collected whole blood samples from 156 donors and performed gene expression analysis of these samples and identified differentially expressed genes (DEGs) in each obese and/or asthma group relative to healthy volunteers. Total mRNA was extracted from peripheral blood using Qiagen RNeasy kit (Qiagen, Valencia, CA) and quantified by NanoDrop (Thermo Fisher Scientific, Waltham, MA). RNA integrity was confirmed using the Agilent 2100 BioAnalyzer (Agilent, Palo Alto, CA). Samples were normalized to the lowest concentration sample, and cDNA was made using Superscript Vilo cDNA synthesis Master Mix (Invitrogen Life Technologies, Grand Island, NY). The cDNA samples were then labeled with biotin with the FL-Ovation cDNA Biotin Module V2 and hybridized to a Human Genome U133 Plus 2.0 Array using a Genechip Hybridization Kit. The microarray chips were washed and stained using a Genechip Hybridization Wash and Stain Kit and then scanned using a Genechip Scanner. All reagents and readers were used according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA). The microarray gene expression data were analyzed using ArrayStudio 7.0 (OmicSoft, Cary, NC). Data from .CEL files from Human Genome U133 Plus 2.0 Affymetrix chips were normalized using robust multiarray averaging and scaled to a mean target intensity of 150. Differentially expressed transcripts were identified by comparing each asthma and obesity group to the non-obese non-asthmatic group.