|
| Status |
Public on Mar 14, 2014 |
| Title |
hMSCs Normoxia_2 |
| Sample type |
RNA |
| |
|
| Source name |
Human bone marrow mesenchymal stem cells 21% Oxygen 24 hrs
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Bone Marrow
diagnosis: Healthy
cell type: Mesenchymal Stem Cells
|
| Treatment protocol |
MSCs were plated at 1×105 cells/cm2 in complete culture medium and incubated under hypoxia (0.5% O2, 5% CO2) or normoxia (21% O2, 5% CO2) for 24 hours using a ProOX Model C21 system (BioSpherix, Redfield, NY, USA).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from normoxic and hypoxic MSCs (3 independent samples each) was extracted using a Kit from Biochain, (Hayward, CA, USA), according to the manufacturer’s instructions. The RNA quality was assessed by formaldehyde agarose gel electrophoresis and quantified using a spectrophotometer (Nanodrop, Wilmington, DE, USA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Raw data were acquired using an Agilent DNA Microarray Scanner and Agilent Feature Extraction Software.
|
| Description |
Healthy donors
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Mar 13, 2014 |
| Last update date |
Mar 14, 2014 |
| Contact name |
Lina A Shehadeh |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Miami
|
| Department |
Medicine
|
| Street address |
1501 NW 10 Ave
|
| City |
Miami |
| State/province |
FL |
| ZIP/Postal code |
33136 |
| Country |
USA |
| |
|
| Platform ID |
GPL14552 |
| Series (1) |
| GSE55875 |
Severe Hypoxia Exerts Parallel and Cell-specific Regulation of Gene Expression and Alternative Splicing in Human Mesenchymal Stem CellsMesenchymal Stem Cells |
|
