gender: M age: 47 site: Van allergen: Orchard Grass mch pc20 (mg/ml) at pre-challenge: 0.28 mch pc20 (mg/ml) at post-challenge: NA % fall in fev1 (early): -23 % fall in fev1 (late): -17 rna integrity number: 8.9 tissue: whole blood
Extracted molecule
total RNA
Extraction protocol
The samples collected pre- and post-challenge were frozen and transported to the laboratory on dry ice, where they were stored at –80°C until RNA extraction. From 8 PAXgene Blood RNA tubes (PreAnalytiX – Qiagen / BD, Valencia, CA, USA), total intracellular RNA was purified from 2.5 mL of whole-blood according to manufacturer protocols using the RNeasy mini kit (Qiagen, Chatsworth, CA, USA). Aliquots of RNA isolated from PAXgene tube samples taken from the 4 individuals were subjected to globin transcript reduction processes using the GLOBINclear™ - Human kit (Ambion – AB, Austin, TX, USA). The yield and quality of RNA was assessed by NanoDrop 8000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA)
Label
biotin
Label protocol
standard Affymetrix protocol
Hybridization protocol
Affymetrix Human Gene 1.0 ST array (Affymetrix, Santa Clara, CA)
Scan protocol
standard Affymetrix protocol
Data processing
Raw data normalization using the 'farms' package consisted of quantile normalization and summarization using factor analysis of probe-level data. Data filtering was also performed using the Informative vs. Non-Informative Calls function in the 'farms' package. Differential gene expression was determined using moderated t-tests in limma using an FDR cut-off of 5%. All statistical analyses were performed in the statistical computing program R.
