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Sample GSM634922 Query DataSets for GSM634922
Status Public on Mar 01, 2012
Title Pig 40 ASC osteogenic differentiation 2 day of differentiation
Sample type RNA
 
Channel 1
Source name ASC at 2 day of osteogenic differentiation
Organism Sus scrofa
Characteristics animal id: Pig 40
tissue: adipose
cell type: ASC Mesenchymal Stem cells
protocol: osteogenic differentiation
time: 2 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

Data table header descriptions
ID_REF
VALUE Lowess normalized log2 ratio (sample/reference)

Data table
ID_REF VALUE
01: A-03 SS00000001 -2.307572802
01: A-05 SS00000002 -0.083141235
01: A-07 SS00000003 2.610700062
01: A-09 SS00000004 3.003961966
01: A-11 SS00000005 3.147306699
01: A-13 SS00000006 0.7031007
01: A-15 SS00000007 0.567545448
01: A-17 SS00000008 1.595980979
01: A-19 SS00000009 0.018634174
01: A-21 SS00000010 -0.089267338
01: A-23 SS00000011 0.14404637
01: C-01 SS00000012 0.552868871
01: C-03 SS00000013 -0.220950447
01: C-05 SS00000014 0
01: C-07 SS00000015 0.604071324
01: C-09 SS00000016
01: C-11 SS00000017 0.075874867
01: C-13 SS00000018 0.292781749
01: C-15 SS00000019 0.119024103
01: C-17 SS00000020 -0.263611599

Total number of rows: 13297

Table truncated, full table size 436 Kbytes.




Supplementary file Size Download File type/resource
GSM634922_OF_40_d2_CY3_082007.gpr_NEW.gpr.gz 2.3 Mb (ftp)(http) GPR
GSM634922_OF_40_d2_CY5_082007.gpr_NEW.gpr.gz 2.4 Mb (ftp)(http) GPR
Processed data included within Sample table

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