|
| Status |
Public on Oct 25, 2008 |
| Title |
Human fibroblast-like cells, biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
The T3DF fibroblast-like cells were derived from hES-T3 cell line and they appeared as flat cells with elongated nucleus and branching pseudopodia.
|
| Organism |
Homo sapiens |
| Characteristics |
Gender: female
Passage: 8
|
| Treatment protocol |
Non
|
| Growth protocol |
hES-T3 cells were transferred into feeder-free and noncoated plate (10 cm) in DMEM supplemented with 10% FBS (GIBCO) under 5% CO2 at 37oC. After 10 Days, cells appeared as fibroblast-like morphology, that is, flat cells with elongated nucleus and branching pseudopodia, and these differentiated fibroblast-like cells are designated as T3DF. These T3DF cells were passaged using trypsin (0.05%, GIBCO) every 4 days or cryopreserved.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions. Total RNAs extracted using TRIZOL reagent were quantified spectrophotometrically. The cDNA synthesis was carried out by using SUPERSCRIPTTM one-step RT-PCR kit (Invitrogen). For the reverse transcription step, the whole 5 ul of the resuspended RNAs were incubated for 60 min at 42oC, then 15 min at 72oC in 50 ul of reaction mixture containing 25 ul of 2x Reaction Mix (Invitrogen) and 1 ul of RT/PLANTINUM Taq Mix (Invitrogen). 28.5 ul of the cDNAs present in the 50 ul RT reaction mixture purified by Microarray Target Purification Kit (Roche Applied Science, Indianapolis, ID, USA, http://www.roche-applies-science.com) were used as templates to amplify cDNA by Microarray Target Amplification Kit. The amplified cDNAs were purified by Microarray Target Purification Kit, and complementary RNAs (cRNAs) were synthesized from 200 ng cDNA with Microarray RNA Target Synthesis Kit (Roche Applied Science).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 micrograms purified cRNA (Expression Analysis Technical Manual, 2004, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 10 micrograms of cRNA were hybridized for 16 hr at 45oC on Affymetrix Human Genome U133 plus 2.0 GeneChip. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
GeneChips from the hybridization experiments were read by the Affymetrix GeneChip scanner 3000.
|
| Description |
The mRNA profiles from the undifferentiated human embryonic stem cell line hES-T3 (T3ES), hES-T3 derived embryoid bodies (T3EB) and hES-T3 differentiated fibroblast-like cells (T3DF) were quantitatively determined.
|
| Data processing |
The data were processed by GeneSpring software version 7.2 GC-RMA preprocessor to use on the CEL files
|
| |
|
| Submission date |
Oct 26, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Sung-Liang Yu |
| E-mail(s) |
slyu@ntu.edu.tw
|
| Phone |
886-2-23958341
|
| Organization name |
National Taiwan University
|
| Department |
Clinical Laboratory Sciences and Medical Biotechnology
|
| Lab |
Microarray Core Facility
|
| Street address |
Jen Ai Road Section1
|
| City |
Taipei |
| ZIP/Postal code |
100 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE9440 |
Expression data from human embryonic stem cell differentiation |
|
| Relations |
| Reanalyzed by |
GSE119087 |
