|
| Status |
Public on Dec 20, 2011 |
| Title |
day 1 adipogenic diff. (miRNA) BR2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Mesenchymal stem cells harvested at day 1 after induction of differentiation
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: primary hMADS
time: day 1 after induction of differentiation
treatment: 10 μg/ml transferrin, 0.86 μM insulin, 0.2 nM of triiodothyronine, 1 μM dexamethasone, 100 μM isobutyl-methylxanthine, and 100 nM rosiglitazone
|
| Treatment protocol |
Two days after confluence (referred to as day 0), cells were cultured in DMEM/Ham's F12 medium supplemented with 10 μg/ml transferrin, 0.86 μM insulin, 0.2 nM of triiodothyronine, 1 μM dexamethasone, 100 μM isobutyl-methylxanthine, and 100 nM rosiglitazone. Three days later, the medium was changed (dexamethasone and isobutyl-methylxanthine were omitted).
|
| Growth protocol |
hMADS cells were grown in Dulbecco's Modified Eagle's Medium (DMEM low glucose) containing 10% fetal calf serum (FCS), and 100 U/ml penicillin and streptomycin. After reaching 80% confluence, adherent cells were detached with 0.25% trypsin EDTA and seeded a density of 4500 cells per cm2. hMADS cells were maintained in proliferation medium supplemented with 2 ng/ml fibroblast growth factor 2 (FGF2)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) or Tri-Reagent (Euromedex, Mundolsheim, France) according to the manufacturer's instructions.
|
| Label |
Cy3,Cy5
|
| Label protocol |
5 µg RNA aliquots of each time point were fluorescently labeled using a miRCURY LNA microRNA Power Labeling Kit (Exiqon, Vedbaek, Denmark) according to manufacturer´s instructions
|
| |
|
| Channel 2 |
| Source name |
Mesenchymal stem cells harvested at day -3 before induction of differentiation
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: primary hMADS
time: day -3 before induction of differentiation
|
| Treatment protocol |
Two days after confluence (referred to as day 0), cells were cultured in DMEM/Ham's F12 medium supplemented with 10 μg/ml transferrin, 0.86 μM insulin, 0.2 nM of triiodothyronine, 1 μM dexamethasone, 100 μM isobutyl-methylxanthine, and 100 nM rosiglitazone. Three days later, the medium was changed (dexamethasone and isobutyl-methylxanthine were omitted).
|
| Growth protocol |
hMADS cells were grown in Dulbecco's Modified Eagle's Medium (DMEM low glucose) containing 10% fetal calf serum (FCS), and 100 U/ml penicillin and streptomycin. After reaching 80% confluence, adherent cells were detached with 0.25% trypsin EDTA and seeded a density of 4500 cells per cm2. hMADS cells were maintained in proliferation medium supplemented with 2 ng/ml fibroblast growth factor 2 (FGF2)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) or Tri-Reagent (Euromedex, Mundolsheim, France) according to the manufacturer's instructions.
|
| Label |
Cy5,Cy3
|
| Label protocol |
5 µg RNA aliquots of each time point were fluorescently labeled using a miRCURY LNA microRNA Power Labeling Kit (Exiqon, Vedbaek, Denmark) according to manufacturer´s instructions
|
| |
|
| |
| Hybridization protocol |
The labeled miRNAs were hybridized on miRNA chips for 16 h at 64 °C in a HS 400 Pro Hybridization Station (Tecan, Grödig, Austria). All hybridizations were repeated with reversed dye assignment (dye-swap).
|
| Scan protocol |
Arrays scanned using the software GENEPIX
|
| Data processing |
Background subtraction as well as GENEPIX global mean and dye swap normalization were applied.
|
| |
|
| Submission date |
May 10, 2011 |
| Last update date |
Dec 20, 2011 |
| Contact name |
Federica Eduati |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Padova
|
| Street address |
Via G. Gradenigo 6/B
|
| City |
Padova |
| State/province |
Italy |
| ZIP/Postal code |
35131 |
| Country |
Italy |
| |
|
| Platform ID |
GPL13514 |
| Series (2) |
| GSE29185 |
Identification of active microRNA/transcription factor feed-forward loops during human adipogenesis (miRNA) |
| GSE29186 |
Identification of active microRNA/transcription factor feed-forward loops during human adipogenesis |
|
