|
| Status |
Public on Nov 10, 2010 |
| Title |
Subject3_EIB_positive_pre_exercise |
| Sample type |
RNA |
| |
|
| Source name |
Induced sputum from EIB positive person
|
| Organism |
Homo sapiens |
| Characteristics |
Induced sputum was conducted with 3% hypertonic saline via an ultrasonic nebulizer (DeVilbiss, Somerset, PA, USA). At 2-min intervals, subjects were asked to clear saliva from their mouth and then expectorate sputum. Sputum was collected over 20 min and was pooled into a single sample container. The induced sputum was placed on ice immediately and processed within 15 min of collection.
|
| Biomaterial provider |
The University of Washington (Seattle, WA).
|
| Treatment protocol |
The University of Washington Institutional Review Board approved the study protocols, and written informed consent was obtained from all participants. Subjects 18-59 years of age were recruited who had a physician diagnosis of asthma for = 1 year, and used only an inhaled beta2-agonist for asthma treatment. In accordance with a priori definitions, asthmatics with a methacholine PC20 = 4 mg/ml were identified with = 20% fall in FEV1 following exercise challenge (EIB+ group) and asthmatic controls without EIB were identified with = 5% fall in FEV1 following exercise challenge (EIB- group). Potential participants were excluded if baseline FEV1 was = 65% predicted, history of smoking cigarettes within the prior year or = 7 pack-year smoking, treated for acute asthma within the prior month, hospitalized for asthma within the prior 3 months, or history of life threatening asthma. Participants were excluded if they had used an inhaled or oral corticosteroid, leukotriene modifier, long-acting antihistamine, cromone, or long-acting beta2-agonist in the 30 days prior to the study. Comparisons were also made to non-asthmatic subjects with negative methacholine (PC20 > 8 mg/ml) and dry air exercise challenge tests (< 20% fall in FEV1 following exercise). Epithelial brushings and endobronchial biopsies were obtained from subjects with asthma and EIB defined by a methacholine PC20 = 4 mg/mL and = 15% fall in FEV1 following exercise challenge. Some epithelial brushings from asthmatics were placed in primary culture and used at passage 1 or 2 for in vitro experiments as detailed in the supplemental material.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The lower airway portion of the induced sputum was selected using a transfer pipette and dispersed in dithiothreitol 0.1% (Calbiochem, La Jolla, CA) in a cool shaking water bath at 20oC for 15 min. A portion of the homogenized sputum was used to assess cell viability, cell count and cell differential. The dispersed induced sputum sample was centrifuged at 250 g for 10 min, the supernatant removed, and the cell pellet was immediately treated with caotropic lysis buffer. Total RNA was extracted using the RNeasy Mini protocol (Qiagen, Valencia, CA).
|
| Label |
biotin
|
| Label protocol |
Total RNA (2 ug) was labeled and hybridized to the Affymetrix GeneChip Human Genome HG-U133A Arrays and hybridized according to manufacurer’s instructions.
|
| |
|
| Hybridization protocol |
The standard hybridization protocol was used as recommended by Affymterix.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneArray Scanner 3000 through the University of Washington Center for Expression Arrays (http://ra.microslu.washington.edu/).
|
| Description |
The lower airway portion of the induced sputum.
|
| Data processing |
Bioconductor GCRMA normalization log2
|
| |
|
| Submission date |
Dec 02, 2008 |
| Last update date |
Sep 05, 2017 |
| Contact name |
James William MacDonald |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Washington
|
| Department |
Environmental and Occupational Health Sciences
|
| Street address |
4225 Roosevelt Way NE
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98105-6099 |
| Country |
USA |
| |
|
| Platform ID |
GPL96 |
| Series (1) |
| GSE13785 |
Novel mediators of eicosanoid and epithelial nitric oxide production in asthma |
|
