|
| Status |
Public on Nov 01, 2005 |
| Title |
ES/N2bFGF_2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Mouse Embryonic Stem Cell
|
| Organism |
Mus musculus |
| Characteristics |
control
|
| Biomaterial provider |
Stem Cell Research Center
|
| Treatment protocol |
no treatment
|
| Growth protocol |
Undifferentiated R1 mouse ES cells (stage 1) were grown and maintained on gelatin-coated dishes with leukemia inhibitory factor (ESGRO, Invitrogen, NY) in ES cell medium, KODMEM supplemented with 15% FBS, nonessential amino acids, beta-mercaptoethanol, L-glutamine, and antibiotics
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared by using TriZol reagent (Invitrogen)
|
| Label |
cy5
|
| Label protocol |
With 5ug of total RNA, Microarray cDNA synthesis kit(Roche) was used for first and second strand cDNA synthesis. Then double strand cDNA was prufied with RNeasy mini kit(Qiagen). For in vitro transcription, T7 MegaScript(Ambion) was used with Cy5-UTP(5mM, Amersham pharmacia) for 16 hours at 37℃.
|
| |
|
| Channel 2 |
| Source name |
N2bFGF
|
| Organism |
Mus musculus |
| Characteristics |
treatment
|
| Biomaterial provider |
Stem Cell Research Center
|
| Treatment protocol |
EBs were dissociated and plated onto tissue culture dishes in the serum-free medium containing Insulin/Transferrin/Selenium/Fibronectin (ITSF). After 6 days, the cells were expanded and plated onto polyornithine (15 mg ml-1) and laminin-coated plates (Becton Dickinson Labware, Bedford, MA) with the N2 medium (Johe, 1996) supplemented with laminin, bFGF (R&D Systems, Minneapolis, MN), murine N-terminal fragment of SHH and murine FGF8 isoform b (R&D Systems) (stage 4) . Finally the cells were induced to differentiated into DA neurons by removal of bFGF.
|
| Growth protocol |
Undifferentiated R1 mouse ES cells (stage 1) were grown and maintained on gelatin-coated dishes with leukemia inhibitory factor (ESGRO, Invitrogen, NY) in ES cell medium, KODMEM supplemented with 15% FBS, nonessential amino acids, beta-mercaptoethanol, L-glutamine, and antibiotics
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared by using TriZol reagent (Invitrogen)
|
| Label |
cy3
|
| Label protocol |
: With 5ug of total RNA, Microarray cDNA synthesis kit(Roche) was used for first and second strand cDNA synthesis. Then double strand cDNA was prufied with RNeasy mini kit(Qiagen). For in vitro transcription, T7 MegaScript(Ambion) was used with Cy3-UTP(5mM, Amersham pharmacia) for 16 hours at 37℃.
|
| |
|
| |
| Hybridization protocol |
the labeled sample was resuspended with Hybridization buffer(50mM Na-Phosphate, ph8.1, 50% Formamide, 6X SSC, 5X Denhardtdts Solution, 0.5% SDS), then hybridized in humidified chamber at 42℃ for 24 hours.
|
| Scan protocol |
Cy3 and Cy5 fluorescent intensities were determined using the GenePix scanner (Axon Instruments), and images were analyzed using the GenePix Pro.
|
| Description |
ES/N2bFGF normalized log ratio
|
| Data processing |
Variance stabilizing normalization was applied with the ‘vsn’ package in Bioconductor using the R statistical package. After performing intensity-dependent global LOWESS regression, spatial and intensity dependent effects were managed by pin-group LOWESS normalization.
|
| |
|
| Submission date |
Oct 28, 2005 |
| Last update date |
Oct 31, 2005 |
| Contact name |
Woong Yang Park |
| E-mail(s) |
[email protected]
|
| Phone |
+82-02-740-8241
|
| Fax |
+82-02-744-4534
|
| Organization name |
Seoul National University
|
| Department |
Biochemistry and Molecular Biology
|
| Lab |
Molecular and Genomic Medicine
|
| Street address |
28 Yongondong, Chongnogu
|
| City |
Seoul |
| ZIP/Postal code |
110-799 |
| Country |
South Korea |
| |
|
| Platform ID |
GPL2987 |
| Series (2) |
| GSE3528 |
Guided differentiation of mouse embryonic stem cell |
| GSE5459 |
Selection of Neural Differentiation-Specific Genes by Comparing Profiles of Random Differentiation |
|
