|
| Status |
Public on May 27, 2013 |
| Title |
Airway_45_6h_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Airway wall_6 hours after physical injury
|
| Organism |
Ovis aries |
| Characteristics |
sheep id: 45
tissue: bronchial wall
sampling protocol: 316
|
| Treatment protocol |
SAMPLING PROTOCOL: Eight sheep were subjected to endobronchial brush biopsies (BBr) on three occasions prior to euthanasia and post mortem examination (PME). The four chosen time points were allocated such that pairs of sheep were exposed to BBr according to one of four possible time point combinations: d3:d1:6h (316), d7:d3:6h (736), d7:d1:6h (716) or d7:d3:d1(731) - with each time-point referring to the interval to PME. At PME, in addition to sampling tissue from sites previously subjected to BBr, additional tissue was harvested from naïve sites not previously exposed to BBr.
Airway wall areas were subjected to endobronchial brush biopsy at intervals prior to euthanasia. At necropsy these areas, as well as an area of the airway wall not subject to biopsy, were identified and each injured airway site was immediately cut into 4 or 5 pieces and those tissues transferred to the RNAlater (Ambion, Inc., Austin, TX) and stored at -80C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from homogenised cartilaginous airway tissue using RNeasy Mini kits (Qiagen, Crawley, West Sussex, UK) following the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
Cy3 labelled cRNA were prepared according to the standard Agilent protocols and kit instructions (Gene Expression Hybridization Kit p/n 5188-5242).
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| |
|
| Hybridization protocol |
Sheep were allocated at random to Agilent slides and samples were allocated at random to arrays within slides so that the potential for any variation between slides or arrays caused by the measurement process biasing the comparison of treatments was negated. cRNA was hybridized for 16 hours at 65°C according to standard protocols (http://www.ark-genomics.org/protocols/).
|
| Scan protocol |
The glass slides containing the RNA samples were scanned using the auto photo multiplier tube (PMT) Axon 4200AL scanner following the manufacturer's standard operation procedures.
|
| Description |
Sample derived at necropsy from an area of airway wall subjected to bronchial brush biopsy 6 hours previously
45_0.25
|
| Data processing |
The microarray expression data was extracted using the Agilent Feature Extraction software version 9.5.3 (protocol GE1-v5_95) to obtain gMeanSignal intensities for each spot on the array - the mean of intensities for each pixel on a spot with no surrogate values inserted for low signal intensities on the grid. Intensities are transformed to logarithms base 2
Normalisation is performed in microarray experiments in an attempt to reduce technical variation (caused by the microarray measurement process) but should preserve biological variation (between animals or conditions). Between slide normalisation in single channel experiments involves stringent assumptions, for example, equal median intensity or equal distribution of intensities (apart from a small number of differentially expressed probes) in all arrays In this experiment, where arrays had samples from different sheep (and would therefore be subject to biological variation) and where initial estimates indicated a large number of differentially expressed probes (around 4000 with overall Timepoints effect statistically significant at 5%), these assumptions seemed inappropriate and between-slide normalisation was not used.
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| |
|
| Submission date |
Apr 06, 2012 |
| Last update date |
May 27, 2013 |
| Contact name |
David Collie |
| E-mail(s) |
david.collie@ed.ac.uk
|
| Organization name |
The Roslin Institute & R(D)SVS
|
| Street address |
Easter Bush Veterinary Centre
|
| City |
Roslin |
| ZIP/Postal code |
EH25 9RG |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL15415 |
| Series (1) |
| GSE37086 |
Gene expression changes associated with the airway wall response to injury |
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