Computational protocol: A Controlled Challenge Study on Di(2-ethylhexyl) Phthalate (DEHP) in House Dust and the Immune Response in Human Nasal Mucosa of Allergic Subjects

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Protocol publication

[…] We performed intraindividual differential (control vs. house dust exposure) gene expression analysis from four subjects of each exposure group. We randomly selected subjects for microarray analysis. The cDNA microarray consisted of 1,232 human genes, 10 different extrahuman spiking controls from Arabidopsis and Sinorhizobium genes, and randomized negative controls (i.e., oligonucleotides that do not bind human mRNA) in 300 and in 12 spot quadruples as described previously (). Briefly, we spotted oligomers (Operon Biotechnologies, Cologne, Germany) on Ultra Gaps 2 coated slides (Corning, Schiphol-Rijk, The Netherlands). We isolated total RNA from biopsy specimens using the RNeasy Mini Kit (Qiagen, Cologne, Germany). For spiking controls, we synthesized synthetic mRNA of 10 different Arabidopsis and Sinorhizobium genes on an ABI 394 synthesizer (Purimex, Staufenberg, Germany). We added 0.2–10 pg of this spiking mRNA to the total RNA of control and exposure specimens, resulting in ratios of 1:2 to 1:10. We reverse transcribed mRNA, labeled it using Superscript-2 (Invitrogen, Karlsruhe, Germany) in combination with the 3 DNA Array 350 Kit (Genisphere, Hatfield, PA, USA), and hybridized it with the spotted oligonucleotides at 57°C overnight. Wescanned microarrays with a dual-laser micro-array scanner (GenePix 4000 B; Axon Instruments, Foster City, CA, USA) recorded with GenePix Pro 4.1 software (Axon Instruments) and carried out further analysis using the platform-independent java application ArrayNorm, version 1.7.2 (). After background subtraction and Lowess subgrid normalization, we performed replicate handling as we averaged quadrupled spots within a slide. Accordingly, we calculated the log2-transformed cyanine 5:cyanine 3 ratio (log2-expression ratio) for all genes and controls. We identified differentially expressed genes between DEHPlow and DEHPhigh exposure using Student’s t-test with Bonferroni step-down correction to adjust the critical alpha limit of 0.05. We considered only genes with valid expression values in each biologic replicate for analysis. We annotated genes according the glossary of the Human Genome Organisation (HUGO) (). The full list of array data is available on the server of the . […]

Pipeline specifications

Software tools GenePix Pro, ArrayNorm
Application Gene expression microarray analysis
Organisms Homo sapiens
Diseases Drug Hypersensitivity
Chemicals Diethylhexyl Phthalate