|
| Status |
Public on Jan 01, 2019 |
| Title |
Donor 3 MSCs, hypoxia treated, with TGFb |
| Sample type |
RNA |
| |
|
| Source name |
bovine bone marrow-derived MSCs preconditioned in hypoxia (2% O2) with TGFb
|
| Organism |
Bos taurus |
| Characteristics |
cell type: bone-marrow derived mesenchymal stem cells
age: juvenile (less than 6 months of age)
tissue: femurs and tibiae
|
| Treatment protocol |
Cells were then passaged and expanded (preconditioned) in basal medium in one of four different conditions for one week: 1) N0: normoxia (21% O2); 2) N1: normoxia + TGF-β3 (10 ng/mL, R&D Systems; Minneapolis, MN); 3) H0: hypoxia (2% O2); 4) H1: hypoxia + TGF-β3. Hypoxic culture was carried out continuously in an environmental workstation (Hypoxystation 35; Hypoxygen, MD, USA) that enabled media changes without exposure to atmospheric conditions.
|
| Growth protocol |
Bone marrow-derived MSCs were isolated from juvenile (age < 6 months) bovine femurs and tibiae. Following the initial isolation, MSCs were expanded to confluence through a single initial passage in standard tissue culture conditions with normal oxygen tension (atmospheric, 21% O2), 5% CO2, in basal medium comprised of high glucose (4.5 g/L) Dulbecco’s Modified Eagle medium (DMEM; ThermoFisher Scientific; Waltham, MA), 10% fetal bovine serum (FBS; ThermoFisher Scientific; Waltham, MA), and 1% penicillin, streptomycin, and fungizone (PSF; ThermoFisher Scientific; Waltham, MA).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from each sample using serial TRIzol (Ambion; Austin, TX)-chloroform extractions, and were in-column treated with RNase-free DNase (Qiagen; Valencia, CA) on miRNeasy columns (Qiagen; Valencia, CA) and eluted following the manufacturer’s protocols.
|
| Label |
biotin
|
| Label protocol |
250ng of total RNA was converted to first-strand cDNA using reverse transcriptase primed by poly(T) and random oligomers that incorporated the T7 promoter sequence. Second-strand cDNA synthesis was followed by in vitro transcription with T7 RNA polymerase for linear amplification of each transcript, and the resulting cRNA was converted to cDNA, fragmented, assessed by Bioanalyzer, and biotinylated by terminal transferase end labeling.
|
| |
|
| Hybridization protocol |
Five and a half micrograms of labeled cDNA were added to Affymetrix hybridization cocktails, heated at 99ºC for 5 min and hybridized for 16 h at 45ºC to Bovine Gene 1.0 ST GeneChips (Affymetrix Inc., Santa Clara CA) using the GeneChip Hybridization oven 645. 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.
|
| Scan protocol |
A GeneChip 3000 7G scanner was used to collect fluorescence signal.
|
| Description |
D5H1
Gene expression data from bovine bone marrow-derived MSCs preconditioned in hypoxia with TGFb
|
| Data processing |
Probe intensities were normalized using RMA as implemented in Partek Genomics Suite (v6.6, Partek, Inc., St. Louis, MO), yielding normalized log2-transformed gene expression intensities.
|
| |
|
| Submission date |
Jan 24, 2018 |
| Last update date |
Jan 01, 2019 |
| Contact name |
Lachlan James Smith |
| Organization name |
University of Pennsylvania
|
| Department |
Department of Orthopaedic Surgery
|
| Street address |
371 Stemmler Hall, 3450 Hamilton Walk
|
| City |
Philadelphia |
| State/province |
Pennsylvania |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL16500 |
| Series (1) |
| GSE109567 |
Expression data from bovine bone-marrow derived MSCs following preconditioning (hypoxia and/or transforming growth factor-beta (TGF-β)) |
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