|
| Status |
Public on May 18, 2007 |
| Title |
repeat 3 hypoxia |
| Sample type |
RNA |
| |
|
| Source name |
peripheral blood lymphocytes incubated at 1 % ambient oxygen for 20 hours.
|
| Organism |
Homo sapiens |
| Characteristics |
lymphocytes isolated from human male 33 years
|
| Treatment protocol |
Peripheral blood lymphocytes were cultured for for 20 hours at 37 degrees celsius under either 20% oxygen or 1% ambient oxygen tensions.
|
| Growth protocol |
Extracted peripheral blood lymphocytes were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Sigma Aldrich lot # 110K8403) and 5mmol of L-glutamine and penicillin/streptomycin antibiotics (Sigma Aldrich).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from approximately 70,000 harvested lyphocytes from each incubation using a column based centrifugation method, designed by Ambion (USA RNAqueous-4PCR cat. # 1914) in accordance with the manufacturers instructions..
|
| Label |
biotin
|
| Label protocol |
Biotin-16 UTP of invitro transcribed RNA. Used Illumina RNA amplification kit ( Cat # 1755 Ambion US).
|
| |
|
| Hybridization protocol |
1500ng of cRNA from each sample was hybridized to each of the six microarrays on the platform for 15 hours at 55 degrees celsius. The microarrays were washed and then stained with streptavidin conjugated to Cy3 fluorescent dye. The staining procedure relies on the affinity of streptavidin to biotin which is incorporated into the UTP residues of the hybridised cRNA sample. Arrays were then washed again and air dried.
|
| Scan protocol |
Microarrays were scanned under 532nm laser illumination using a confocal type image system (BeadArray Reader, Illumina California USA). Each sequence specific probe is represented on average by 30 beads on each microarray. Fluorescence intensities from each bead were determined and each specific probe signal was automatically calculated by BeadStudio software (Illumina California) using a trimmed average of the corresponding replicate bead signals. Background intensities were calculated and subtracted from each data point using ~ 700 negative control probes on each array.
|
| Description |
na
|
| Data processing |
Once the average signal intensity from each gene specific probe on the microarray had been determined, a small offset was added to each data point to reduce variability at low intensity levels and the data was normalized using the quantile normalisation method contained in the affy package (Gautier et al., 2004) on the R statistical analysis platform (www.r-project.org). The reduction in variability at low intensities is obtained at the expense of slighter lower differential expression values.
The data were then reviewed for gene expression and hypoxic regulation using the Genespring analysis platform (Agilent technologies USA). Statistical analysis was performed using the limma library from the bioconductor repository (www.bioconductor.org) running on the R-platform. A multiple testing correction was applied using the limma algorithm that controls for the false discovery rate. The three repeat cRNA samples hybridised to the six microarrays were considered as a paired set of hybridisations. The gene specific signal intensities in the normoxic cRNA samples were compared to that of the hypoxic cRNA samples. Ratios of the average signal intensities under hypoxia compared to normoxia were calculated for each transcript and the mean values of the signal intensities from the three repeated normoxic/hypoxic exposures were compared using the limma software package to perform a paired study design. The average signal intensities, the average hypoxia to normoxia expression ratios (for the three paired normoxic/hypoxic lymphocyte incubations) and the p values representing the significance of change of expression in hypoxic conditions for all of the transcripts assayed for on the Illumina Sentrix Human 6-Expression BeadChip platform are submitted as a Series table.
|
| |
|
| Submission date |
May 17, 2007 |
| Last update date |
May 17, 2007 |
| Contact name |
Jerome Tremblay Brooks |
| E-mail(s) |
[email protected]
|
| Phone |
961410862
|
| Organization name |
University of Oxford
|
| Department |
Department of Physiology Anatomy and Genetics
|
| Lab |
Peter Robbins
|
| Street address |
Parks Road
|
| City |
Oxford |
| State/province |
Oxon |
| ZIP/Postal code |
OX1 3JA |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL2507 |
| Series (1) |
| GSE7833 |
Regulation of mRNA transcript expression by hypoxia in human peripheral blood lymphocytes |
|
