|
| Status |
Public on Jul 31, 2010 |
| Title |
Wild_type_Drosophila_normobaric_hypoxia_sample5 |
| Sample type |
RNA |
| |
|
| Source name |
DmWT CH # 5, normobaric hypoxia, two weeks
|
| Organism |
Drosophila melanogaster |
| Characteristics |
id: Sample # 3
age: Adult (3-5 days old)
strain: Wild type
treatment: Exposed to normobaric hypoxia at Philadelphia-PA for two weeks.
|
| Treatment protocol |
In order to understand the chronic hypoxia (CH) effect upon the absence of dystrophin, Drosophila melanogaster wild type and the model for DMD (dmDys), in which all dystrophins expression was knocked out by iRNA, were exposed to high altitude hypoxia (hypobaric hypoxia) during a 16-day climbing period reaching the summit of Mount McKinley (6194 meters above sea level). Furthermore, dmDys and Drosophila wild type were exposed to normobaric hypoxia (hypoxic chamber) following the same oxygen levels observed during the climbing expedition and to normoxic conditions for comparison.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated and purified from each fly by RNeasy micro kit (Qiagen, Valencia, CA) as described by manufacturer.
|
| Label |
Biotin
|
| Label protocol |
100ng of total RNA was converted to first-strand cDNA using reverse transcriptase primed by a poly(T) oligomer that incorporated a synthetic RNA sequence. Second-strand cDNA synthesis was followed by ribo-SPIA (Single Primer Isothermal Amplification, NuGEN Technologies Inc. San Carlo, CA) for linear amplification of each transcript, and the resulting cDNA was fragmented, assessed by Bioanalyzer, and biotinylated
|
| |
|
| Hybridization protocol |
cDNA yields ranged from 5000-6000ug, and 3.75ug were added to Affymetrix hybridization cocktails, heated at 99ºC for 2 min and hybridized for 16 h at 45ºC to Drosophila V2.0 GeneChips (Affymetrix Inc., Santa Clara CA). The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain. A confocal scanner was used to collect fluorescence signal after excitation at 570 nm.
|
| Scan protocol |
The chip was scanned at 6um resolution with Agilent model G2500A GeneArray scanner. A visual quality control measurement was performed to ensure proper hybridization after each chip was scanned.
|
| Description |
Each sample was made using single whole fly.
|
| Data processing |
Quality control parameters such as scaling factors used to normalize the chips, average background, and noise were also evaluated. In addition, raw intensities for each probe set were stored in electronic formats by the GeneChip Operating System version 1.1 (GCOS1.1, Affymetrix Inc.) for GC-RMA analysis in GeneSpring Software later on.
|
| |
|
| Submission date |
Apr 28, 2009 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Matias Mosqueira |
| E-mail(s) |
[email protected]
|
| Phone |
+496221544143
|
| Organization name |
Heidelberg University Hospital
|
| Department |
Institute of Physiology and Pathop
|
| Lab |
Cardio-Ventilatory Muscle Physiology
|
| Street address |
Im Neuenheimer Feld, 326 R414
|
| City |
Heidelberg |
| State/province |
Baden-Wuttenberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
| |
|
| Platform ID |
GPL1322 |
| Series (1) |
| GSE15879 |
Effects of chronic hypoxia on Duchenne Muscular Dystrophy Drosophila melanogaster model |
|
| Relations |
| Reanalyzed by |
GSE119084 |
