|
| Status |
Public on Sep 20, 2012 |
| Title |
DOX.IL1.4h.4 |
| Sample type |
RNA |
| |
|
| Source name |
Pdx1 overexpression, Il-1b added, harvest after 4h, replica 4
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell line: rat insulinoma INS-1ab cell line with doxycyclin inducible Pdx1 expression
agent: 500 ng/ml doxycyclin
agent: not added
time point: 4h
biological replica: 4
|
| Treatment protocol |
After 24 h, 40 ng/ml IL-1b was added, and cells were harvested at 2h, 4h, 6h, 12h, and 24h, respectively.
|
| Growth protocol |
Cells were cultured in RPMI 1640 with or without 500 ng/ml doxycycline.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Isolation of total cellular RNA was performed with cells from a single 10cm diameter dish. The monolayer was rapidly washed twice with phosphate-buffered saline, pH 7.5, and lysed in a guanidinium thiocyanatesolution. The resulting suspension was homogenized by several passes through a 21-gaugeneedle. Phenol extraction of the homogenate and isopropyl alcohol precipitation of RNA were performed. Two additional cycles of suspension in the guanidinium thiocyanate solution and isopropyl alcohol precipitation were carried out.
|
| Label |
biotin
|
| Label protocol |
Total RNA was processed following the GeneChip Expression 30-Amplification Reagents One-cycle cDNA synthesis kit instructions (Affymetrix Inc, Santa Clara, CA, USA) to produce double-stranded cDNA. This was used as a template to generate biotin-labelled cRNA following manufacturer’s specifications.
|
| |
|
| Hybridization protocol |
Fifteen micrograms of biotin-labelled cRNA was fragmented and 10 mg were hybridized onto Affymetrix Rat Genome 230 2.0 arrays overnight in the GeneChip Hybridisation oven 6400 using standard procedures, The arrays were washed and stained in a GeneChip Fluidics Station 450.
|
| Scan protocol |
Scanning was carried out using the GeneChip Scanner 3000 and image analysis was performed using GeneChip Operating Software.
|
| Data processing |
GCRMA
|
| |
|
| Submission date |
Sep 06, 2012 |
| Last update date |
Sep 20, 2012 |
| Contact name |
Morten Tonnesen |
| E-mail(s) |
mtnt@novonordisk.com
|
| Organization name |
Hagedorn Research Institute
|
| Street address |
Niels Steensensvej 1
|
| City |
Gentofte |
| ZIP/Postal code |
2820 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL1355 |
| Series (1) |
| GSE40642 |
Pdx1 overexpression sensitizes to IL-1b-induced apoptosis |
|
