|
| Status |
Public on Oct 29, 2009 |
| Title |
3FP1 |
| Sample type |
RNA |
| |
|
| Source name |
hMSCS
|
| Organism |
Homo sapiens |
| Characteristics |
passage: 1
medium: FGF supplemented
|
| Treatment protocol |
From the first medium change (day 4), some of the plates were assigned to receive control medium, while the rest of the plates received medium supplemented with 10 ng/ml of rhFGF-2. This dose was chosen based on previous studies. Cultures were fed twice per week
|
| Growth protocol |
Bone marrow aspirates were washed with control medium consisting in low glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum from a selected lot (DMEM-LG + 10% FBS) and subjected to a pre-formed Percoll density gradient to isolate mononucleated cells. The mononucleated cells were washed with control medium and seeded at a density of 1.8 x 105 cells/cm2 in control medium to establish primary cultures of human bone marrow-derived MSCs. All cell-culture was done at 37°C in a humidified atmosphere of 95% air and 5% CO2. In order to keep their growth at an exponential rate and prevent spontaneous differentiation or loss of differentiation potential, hMSCs must be subcultured before the cells become confluent. Typically, they were passaged when the cultures were 80 - 90% confluent. Primary cultures were usually subcultured around day 14 (± 3 days). Subsequently, the cells were subcultured approximately every 7 (± 2) days. Plates assigned to the different study groups were subcultured at the same time which resulted in different levels of confluence in the treatment groups as result of the previously reported differences in cell proliferation and cell size.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Five to eight µg of total RNA were converted into double stranded cDNA using Superscript II reverse transcriptase (Gibco BRL, Rockville, MD) and an oligo-dT primer with a T7 RNA polymerase promoter linked to its 5’ end. cDNA was cleaned and used in an in vitro transcription (IVT) reaction to generate adequate amplified, biotin-labeled cRNA (50-80 µg).
|
| |
|
| Hybridization protocol |
cRNA was then fragmented, and 15 µg added to 300 µl hybridization cocktail. The hybridization cocktail is a multi-component, buffered environment (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween 20; 0.1 mg/ml Herring Sperm DNA, 0.5 mg/ml acetylated BSA, and Affymetrix IVT controls, as per manufacturer). Four IVT products of bacterial genes were added at final concentrations ranging from 1.5-100 pM. Spiked transcripts served as hybridization controls and were always monitored as part of our quality control regimen. Fragmented biotin-labeled cRNA was added, and the final volume is adjusted to 300 µl with molecular grade water. Samples were hybridized onto Affymetrix (Santa Clara, CA) H-U133A 2.0 human microarrays
|
| Scan protocol |
The manufacturer’s standard post-hybridization wash, double-stain, and scanning protocols use an Affymetrix GeneChip Fluidics Station 400 and a Hewlett Packard Gene Array scanner
|
| Description |
Gene expression data from culture-expanded mesenchymal stem cells
|
| Data processing |
Raw data from microarray scans were initially normalized and analyzed with Affymetrix Microarray Suite (MAS) v.5.0
|
| |
|
| Submission date |
Feb 13, 2009 |
| Last update date |
Aug 13, 2015 |
| Contact name |
Luis A Solchaga |
| E-mail(s) |
las29@case.edu
|
| Organization name |
Case Western Reserve University
|
| Street address |
2103 Cornell Road
|
| City |
Cleveland |
| State/province |
OH |
| ZIP/Postal code |
44106 |
| Country |
USA |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE14828 |
FGF-2 ENHANCES PROLIFERATION AND DELAYS LOSS OF CHONDROGENIC POTENTIAL IN HUMAN ADULT BONE MARROW-DERIVED MSCs |
|
