|
| Status |
Public on Nov 21, 2008 |
| Title |
Lung cells_24hrHypoxia_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Lung Microvascular endothelial cells,hypoxia,24hr
|
| Organism |
Homo sapiens |
| Characteristics |
Primary cells
|
| Treatment protocol |
At experiment start, medium was changed and dishes were transferred to the hypoxia chamber (Coy Labs, USA) and cultured in an atmosphere of 1% O2, 5% CO2 and 94% N2 for 3hr, 24hr or 48hrs. Control conditions were achieved by culture in 21% O2, 5% CO2 and 74% N2 in a cell-culture incubator.
|
| Growth protocol |
Primary human microvascular endothelial cells from lung and cardiac tissue were grown on sterile tissue culture dishes in Endothelial Growth Medium (EGM-2MV; Code: CC-3202) according to the manufacturers instructions (Lonza Bioscience formerly Cambrex, UK).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At experiment end, cell medium was removed, cells washed with PBS and thoroughly lysed in RLT buffer (RNeasy Mini kit, Qiagen, UK). Total RNA extraction, with on-the-column DNase treatment, was carried out using RNeasy Mini Kits (Qiagen Ltd, UK) according to the maunfacturer's instructions.
|
| Label |
Biotin
|
| Label protocol |
Total RNA (5ug) was reverse transcribed to cDNA (one-cycle cDNA synthesis kit), clean up of double stranded cDNA, biotin labelling of anti-sense cRNA (integrity checked on the Bioanalyzer 2100, Agilent technologies, USA) and clean up and fragmentation of biotinylated cRNA were all carried out according to the manufacturers protocol (Affymetrix, USA).
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| |
|
| Hybridization protocol |
Fragmented cRNA (15ug) was hybridised to chips for 16 hrs at 45ºC in a hybridisation oven (AffyMetrix, USA) with rotation at 60rpm. Immediately following hybridisation, the array underwent automated washing and staining protocol on the automatic fluidics station 400, (AffyMetrix, USA).
|
| Scan protocol |
Arrays were scanned on a GeneChip 3000 scanner (AffyMetrix, USA).
|
| Description |
All cells used in these experiments were passage 6-7 and were routinely checked for mycoplasma contamination using the VenorGeM PCR kit (Cambio Ltd, UK.).
|
| Data processing |
Affymetrix .cel files were imported and normalised using algorithms of GeneSpring 6.0 software i.e. data was normalised by applying the following data transformation criteria: a) measurements less than 0.01 were set to 0.01, b) per chip: normalised to 50th percentile and c) per gene: normalise to median. Genes that were absent in a majority of chips were eliminated from further analyses.
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| |
|
| Submission date |
May 05, 2008 |
| Last update date |
Nov 21, 2008 |
| Contact name |
Christine Marie Costello |
| Phone |
00353 01 7166738
|
| Fax |
00353 01 7166892
|
| Organization name |
Conway Institute
|
| Street address |
University College Dublin
|
| City |
Dublin |
| ZIP/Postal code |
Dublin 4 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL96 |
| Series (1) |
| GSE11341 |
Lung selective gene responses to alveolar hypoxia |
|
