|
| Status |
Public on Aug 03, 2015 |
| Title |
FB3136_0m_5 |
| Sample type |
RNA |
| |
|
| Source name |
primary fibroblast, serum starved
|
| Organism |
Homo sapiens |
| Characteristics |
individual id: FB3136
cell type: primary fibroblast
agent: no insulin
time point (insulin treatment): baseline
|
| Treatment protocol |
To minimize technical variability, fibroblasts from the 35 individuals were passaged, treated and harvested in one batch for the signaling assays and microarray experiments. Fibroblasts from 35 individuals were serum starved for 18 hours and treated with 100nM insulin for one hour and 6 hours. In the ERK inhibition studies, cells from 4 individuals were treated with 10 uM UO126 (Cell Signaling) or DMSO (Sigma-Aldrich) for 1 hour before 100nM insulin or ddH2O (mock treatment) was added, and cells were incubated for another 1 hour or 6 hours before being harvested.
|
| Growth protocol |
Primary fibroblasts were cultured at 37°C in 5% CO2 in MEM medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/ml streptomycin, and were passaged every 3 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells before or after treatment using RNeasy Micro Kit (QIAGEN).
|
| Label |
biotin
|
| Label protocol |
500ng of total RNA was used for each sample. cRNA was amplified and biotin-labeled following manufacturer's protocol using MessageAmp™ Premier RNA Amplification Kits (Ambion).
|
| |
|
| Hybridization protocol |
cRNA was fragmented in 1X Fragmentation buffer (Ambion). 10 ug of cRNA were hybridized for 16 hours at 45C on GeneChip Human Genome HG-U133A_2 Arrays (GEO Platform GPL571). GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G.
|
| Description |
primary fibroblast, serum starved
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.
|
| |
|
| Submission date |
May 18, 2010 |
| Last update date |
Aug 03, 2015 |
| Contact name |
Isabel Xiaorong Wang |
| Organization name |
HHMI/University of Michigan
|
| Department |
Pediatrics&Genetics
|
| Lab |
Dr. Vivian G. Cheung Lab
|
| Street address |
210 washtenaw ave
|
| City |
Ann Arbor |
| State/province |
Michigan |
| ZIP/Postal code |
48109 |
| Country |
USA |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE21891 |
Expression data from insulin-treated human primary fibroblasts and effects of U0126 on insulin-induced gene expression |
|
