|
| Status |
Public on Mar 01, 2012 |
| Title |
Pig 40 BMSC adipogenic differentiation 21 day of differentiation |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
BMSC at 21 day of adipogenic differentiation
|
| Organism |
Sus scrofa |
| Characteristics |
animal id: Pig 40
tissue: bone
cell type: BMSC Mesenchymal Stem cells
protocol: adipogenic differentiation
time: 21 days
|
| Treatment protocol |
Adipose and bone marrow derived stem cells were induced to differentiate towards bone and adipose with specific differentiation media. The medium for the osteogenic differentiation consisted of high glucose DMEM supplemented with 100 nM dexamethasone, 10 mM b-glycerophosphate, 0.05 mM ascorbic acid-2-phosphate, 10% FBS, 1% Penicillin G-Streptomycin, and 5.0 mg/L Amphotericin B. The adipogenic medium consisted of basic high glucose DMEM supplemented with 1.0 mM dexamethasone, 0.5 mM isobutylmethylxanthine, 10 mM insulin, 200 mM indomethacin, 10% FBS, 1% Penicillin G-Streptomycin, and 5.0 mg/L Amphotericin B.
|
| Growth protocol |
Cells were plated in 75 cm2 Corning cell culture flasks at 7.5 X 105 cells in 15 mL of culture medium and incubated at 39°C and 5% CO2 in 100% humidified air. The culture medium used was high glucose Dulbecco's Modified Eagle's Medium supplemented with 10% Fetal Bovine Serum, antibiotic and antimycotic. Medium was changed every other day and the cells were maintained in culture for 3 passages before starting the adipogenic and osteogenic differentiation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At 0, 2, 7, and 21 days of differentiation cells were trypsinized and total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Any residual genomic DNA was eliminated by treatment with RNase-Free DNase Set (Qiagen). RNA concentration was measured with a NanoDrop ND-1000 spectrophotometer.
|
| Label |
Cy3,Cy5
|
| Label protocol |
Labelled with Cy3 and Cy5 during cDNA synthesis from total RNA using Superscript III Kit (Invitrogen).
|
| |
|
| Channel 2 |
| Source name |
reference
|
| Organism |
Sus scrofa |
| Characteristics |
reference composition: A mixture of total RNA from 4 porcine tissues (32% liver, 32% jujenum, 24% kidney, and 10% mammary tissue)
|
| Treatment protocol |
Adipose and bone marrow derived stem cells were induced to differentiate towards bone and adipose with specific differentiation media. The medium for the osteogenic differentiation consisted of high glucose DMEM supplemented with 100 nM dexamethasone, 10 mM b-glycerophosphate, 0.05 mM ascorbic acid-2-phosphate, 10% FBS, 1% Penicillin G-Streptomycin, and 5.0 mg/L Amphotericin B. The adipogenic medium consisted of basic high glucose DMEM supplemented with 1.0 mM dexamethasone, 0.5 mM isobutylmethylxanthine, 10 mM insulin, 200 mM indomethacin, 10% FBS, 1% Penicillin G-Streptomycin, and 5.0 mg/L Amphotericin B.
|
| Growth protocol |
Cells were plated in 75 cm2 Corning cell culture flasks at 7.5 X 105 cells in 15 mL of culture medium and incubated at 39°C and 5% CO2 in 100% humidified air. The culture medium used was high glucose Dulbecco's Modified Eagle's Medium supplemented with 10% Fetal Bovine Serum, antibiotic and antimycotic. Medium was changed every other day and the cells were maintained in culture for 3 passages before starting the adipogenic and osteogenic differentiation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At 0, 2, 7, and 21 days of differentiation cells were trypsinized and total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Any residual genomic DNA was eliminated by treatment with RNase-Free DNase Set (Qiagen). RNA concentration was measured with a NanoDrop ND-1000 spectrophotometer.
|
| Label |
Cy5,Cy3
|
| Label protocol |
Labelled with Cy3 and Cy5 during cDNA synthesis from total RNA using Superscript III Kit (Invitrogen).
|
| |
|
| |
| Hybridization protocol |
Same amount of labelled cDNA from sample and reference were hybridised using 80 μL of the hybridization buffer #1 (Ambion). Slides placed in humidified slide chambers (Corning) in water bath for 40 h at 45 °C. Prior scanning the microarray were washed for 5 min by agitation 3 times with wash buffers in the following order: 1×SSC and 0.2% SDS solution preheated at 42°C, 0.1×SSC and 0.2% SDS solution, and 0.1×SSC solution. Lastly, slides were inserted into a 50 mL tube, spin-dried and gassed with Argon to preserve dye from bleaching.
|
| Scan protocol |
Arrays were scanned with a ScanArray 4000 (GSI-Lumonics) dual-laser confocal scanner and images were processed and edited using GenePix 6.0 (Axon Instruments).
|
| Description |
A mixture of total RNA from 4 porcine tissues (32% liver, 32% jujenum, 24% kidney, and 10% mammary tissue) were used as reference (Cy5 in Ch1 and Cy3 in Ch2)
|
| Data processing |
Array quality was assessed using an in-house parser written in Perl language. Spots that received a -100 flag by GenePix 6.0 were removed from further analysis and background intensity was subtracted from the foreground intensity. Spots on the slide were considered “good” if the median intensity of sample was ≥3 standard deviation above median background for each channel (i.e., dye). Spots were flagged “present” when both dyes passed the criteria, “marginal” if only one dye passed the criteria, or “absent” when both dyes failed to pass the criteria. Statistical analysis was conducted on oligos that were flagged as “present” and “marginal”. Data from a total of 82 microarrays were normalized for dye and array effects (i.e., Lowess normalization and array centering and scaling) before statistical analysis.
|
| |
|
| Submission date |
Dec 05, 2010 |
| Last update date |
Mar 01, 2012 |
| Contact name |
Massimo Bionaz |
| E-mail(s) |
massimo.bionaz@oregonstate.edu
|
| Phone |
5417379507
|
| Organization name |
Oregon State University
|
| Department |
Animal and Rangeland Sciences
|
| Lab |
Massimo Bionaz
|
| Street address |
316A Withycombe Hall
|
| City |
Corvallis |
| State/province |
OR |
| ZIP/Postal code |
97331 |
| Country |
USA |
| |
|
| Platform ID |
GPL9710 |
| Series (1) |
| GSE25854 |
Transcriptomics adaptation of porcine adipose- and bone marrow-derived stem cells during adipogenic and osteogenic differentiation |
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