|
| Status |
Public on Sep 17, 2008 |
| Title |
RPTEC_normoxic_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
renal proximal tubule epithelial cells, normoxic
|
| Organism |
Homo sapiens |
| Characteristics |
Organism Part: kidney Gender: female Age: 55 Disease state: normal tissue
|
| Biomaterial provider |
BioWhittaker/Lonza
|
| Treatment protocol |
Passage 4 renal proximal tubule epithelial cells were cultured for 24h in a humidified
atmosphere of 5% CO2 and 95% air.
|
| Growth protocol |
Human renal proximal tubule epithelial cells were maintained in REGM with supplements and growth factors (REGM BulletKit CC-3190; Lonza) according to the provider’s instructions. Seeding density for subculturing was 2500 cells per square cm. The cells were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using TRIZOL reagent (Invitrogen) and purified on RNeasy Miniprepcolumns (Qiagen) according to the manufacturers' recommendations.
|
| Label |
Biotin
|
| Label protocol |
10 µg of total RNA was reverse transcribed using the SuperScript Choice system for cDNA synthesis (Invitrogen) according to the protocol recommended by Affymetrix. Theoligonucleotide used for priming was 5'-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24-3' (Genset Oligo). Double-stranded cDNA was cleaned by phenol:chloroform extraction and the
aqueous phase removed by centrifugation through Phase-lock Gel (Eppendorf). In vitro transcription was performed on 1 µg of cDNA using the Enzo BioArray High Yield RNA transcript labelling kit (Enzo Diagnostics). The cRNA was cleaned using RNAeasy clean-up columns (Qiagen). The cRNA was fragmented by heating in 40 mM Tris-acetate pH 8.1, 100mM KOAc, 30 mM MgOAc.
|
| |
|
| Hybridization protocol |
10 µg of fragmented cRNA were hybridised (45°C, 16 hours). Hybridization was controled by use of the GeneChip Eukaryotic Hybridization Control Kit (Affymetrix).
|
| Scan protocol |
Washing and staining was performed in a Fluidics Station 400 (Affymetrix) using the
protocol EukGE-WS2v4 and scanned in an Affymetrix GeneChip scanner. Chip analysis was performed using the Affymetrix Microarray Suite v5.
|
| Description |
Three independent biological replicates were performed.
|
| Data processing |
The microarray CEL-files (Affymetrix HGU133A platform) were read into the statistical software “R” [1], where all subsequent data analysis was done. Expression index computation was calculated using the RMA algorithm with default settings (i.e. “RMA” background correction, “quantiles” normalisation, “pmonly” Perfect Match correction, and “medianpolish” probe set summary calculation). Gene expression levels in Control and hypoxia-treated samples were compared by standard t-tests, and the p-values were corrected for multiple testing by controlling the False Discovery Rate (FDR). The FDR was set to 5% - meaning that around 5% of the differentially expressed genes/probe sets can be expected to be false positives. Along with basic annotation the data for the differentially expressed genes were collected in an Excel file.
[1]: R Development Core Team (2007). R: A language and environment for
statistical computing. R Foundation for Statistical Computing,
Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org.
|
| |
|
| Submission date |
Sep 16, 2008 |
| Last update date |
Sep 16, 2008 |
| Contact name |
Peter Staller |
| E-mail(s) |
[email protected]
|
| Phone |
+4535325682
|
| Fax |
+4535325669
|
| Organization name |
University of Copenhagen
|
| Department |
BRIC
|
| Street address |
Ole Maaløes Vej 5
|
| City |
Copenhagen |
| ZIP/Postal code |
2200 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL96 |
| Series (1) |
| GSE12792 |
Renal Proximal Tube Epithelial Cells at 24h and 1% oxygen |
|
