|
| Status |
Public on Dec 20, 2011 |
| Title |
day 15 adipogenic diff. (mRNA) BR1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Mesenchymal stem cells harvested at day 15 after induction of differentiation
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: primary hMADS
time: day 15 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 then dexamethasone and sobutyl-methylxanthine omitted at day 3
|
| 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 at 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 |
15 µg RNA aliquots of each time point were fluorescently labelled with Cy3 or Cy5 during cDNA synthesis from total RNA using Superscript II Kit (Invitrogen) as manufacturers instructions. Samples purified using QIAGEN PCR purification kit before hybridization.
|
| |
|
| 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 at 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 |
15 µg RNA aliquots of each time point were fluorescently labelled with Cy3 or Cy5 during cDNA synthesis from total RNA using Superscript II Kit (Invitrogen) as manufacturers instructions. Samples purified using QIAGEN PCR purification kit before hybridization.
|
| |
|
| |
| Hybridization protocol |
The labeled cDNA was hybridized on human oligo chips in a water bath for 16h at 42°C. Arrays were then washed in 2x SSC/ 0.1% SDS, 1x SSC, and 0.5x SSC, each at 42°C for 5 min prior to drying and scanning.
|
| Scan protocol |
Arrays scanned using the AXON 4000B scanner and the GENEPIX software
|
| 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 |
GPL13513 |
| Series (2) |
| GSE29182 |
Identification of active microRNA/transcription factor feed-forward loops during human adipogenesis (mRNA) |
| GSE29186 |
Identification of active microRNA/transcription factor feed-forward loops during human adipogenesis |
|
