|
| Status |
Public on Mar 17, 2011 |
| Title |
KO-control-5 |
| Sample type |
RNA |
| |
|
| Source name |
Myd88 KO control tissue 5
|
| Organism |
Mus musculus |
| Characteristics |
strain: myd88 KO
weight: 25-30g
age: 4 month
gender: n/a
tissue: whole trachea
|
| Biomaterial provider |
Dr Adam Giangreco, University College London
|
| Treatment protocol |
Mice were used as control or exposed to intratracheal 2% polidocanol 3 days prior to collection (injury). Mice were culled by pentobarbitol overdose and exsanguinated. Whole trachea was isolated, excess connective tissue removed, and immediately processed for RNA purification
|
| Growth protocol |
Mice were maintained on a reversed 12:12-h light-dark cycle with controlled temperature (21 ± 1°C) and humidity. Food and water were provided ad libitum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the Qiagen RNeasy kit. The integrity of the RNA was confirmed with analysis by the Agilent 2100 bioanalyser using the RNA 6000 LabChip kit.
|
| Label |
Cy3
|
| Label protocol |
600 ng RNA and 3 µl Agilent One-Color RNA Spike-In RNA were labelled with the Agilent Low RNA Input Linear Amplification Kit PLUS, One-Color according to Manufacture’s instructions as follows: 1.2 µl T7 Promoter Primer was added to 600 ng RNA and 3 µl spike in control and denatured at 65 ºC. First Strand Buffer, DTT, dNTP MMLV and RNaseOut was added. The cDNA was synthesised during the following incubation step (2h at 40 ºC). After 10 min denaturation at 65 ºC and the addition of Cy-labelled CTP, Transcription Buffer, DTT, NTP, PEG, RNaseOUT, Inorganic Phosphatase, and T7 RNA Polymerase the synthesis of the fluorescent labelled cRNA was performed during the second incubation step (2 h at 40 ºC). The labelled cRNA was purified with the Qiagen RNeasy Mini Kit according to Manufacturer’s protocol.
|
| |
|
| Hybridization protocol |
The Agilent Hybridisation Kit (catalogue number 5188-5242) was used in conjunction with Agilent Mouse Oligo Arrays (catalogue number G4122F). 2μg of the labelled sample RNA were used for hybridisation according to the Agilent One-Color Microarray-Based Gene Expression Analysis Protocol The hybridisation was performed for 17 h at 65 ºC at 10 rpm. Slides were them washed for 1 min at 22 ºC in Wash Solution 1 (catalogue number 5188-5325) and 1 min at 22 ºC in Wash Solution 2, pre-warmed to 37 ºC (catalogue number 5188-5326). Slides were incubated for 30s in Agilent Stabilisation and Drying Solution (catalogue number 5185-5979).
|
| Scan protocol |
Microarrays were scanned using the Agilent Technologies Scanner G2505B US45102871 (ChipScan software version A.7.0.1), scan region 61 X 21.6mm, scan resolution 5 micron single pass, extended dynamic range selected (XDR Hi 100%; XDR Lo 10%).
|
| Description |
none
|
| Data processing |
Data was extracted from the scanned image using the Agilent Feature Extraction Software version. 9.1.3.1 (protocol GE1-v5_91_0806). The VALUE column came from the gProcessedSignal column of the result table.
|
| |
|
| Submission date |
Jul 23, 2009 |
| Last update date |
Mar 17, 2011 |
| Contact name |
Adam Giangreco |
| E-mail(s) |
a.giangreco@ucl.ac.uk
|
| Organization name |
UCL
|
| Department |
Respiratory Research
|
| Lab |
Sam Janes
|
| Street address |
5 University Street
|
| City |
London |
| ZIP/Postal code |
WC1E 6JJ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE17268 |
Polidocanol injury wildtype versus Myd88 KO trachea |
|
