|
| Status |
Public on Jun 24, 2010 |
| Title |
GM4DC_LPS+IFNg_20hrs_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
GM + IL4 Dendritic cells exposed to LPS + IFNg
|
| Organism |
Mus musculus |
| Characteristics |
gender: female
age: 6-8 weeks
cells: dendritic cells from bone marrow
strain: NOD (non-obese diabetic)
|
| Biomaterial provider |
Morel Lab
|
| Treatment protocol |
GM and GM4 DC (10^6/ml) were cultured in the presence of LPS (10 µg/ml) (Sigma) and IFN-γ (50 ng/ml) (BD PharMingen). Cells were harvested at 0, 6 and 20 hours after activation and total RNA was isolated according to Qiagen’s protocol (Qiagen Inc). Each time point was replicated 2-3 times; thus a total of 15 microarrays were used for the current analysis.
|
| Growth protocol |
BM cultured for 4 days in GM-CSF (1ng/ml) + IL-4 (1ng/ml). DC purified on 14.5% metrizamide gradient.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen kit
|
| Label |
Biotin
|
| Label protocol |
ENZO BioArray high efficiency RNA transcript labeling kit
|
| |
|
| Hybridization protocol |
The resulting biotinylated cRNA was cleaned up using an Affymetrix RNA clean up column. An aliquot of 20 micrograms of cRNA was incubated at 94oC in fragmentation buffer for 35 minutes to break the RNA into segments of 35 to 200 bases. A 1μl aliquot of the sample was run on an Agilent Bioanalyzer to verify that fragmentation had resulted in RNA of the desired size distribution.
Fifteen micrograms of the fragmented RNA was added to a final volume of 300 μl hybridization cocktail (Affymetrix). An appropriate volume of this sample was applied to the Affymetrix MG U74Av2 gene chip and the chip was incubated overnight at 45oC with rotation. Following hybridization the sample was removed and the GeneChip cassette filled with non-stringent wash buffer. The chips were loaded onto an Affymetrix Fluidics station for wash and stain.
|
| Scan protocol |
Samples were scanned using the GeneArray® scanner.
|
| Description |
The RNA labeling and microarray analysis was performed by the PittArray Facility
|
| Data processing |
Affymetrix MAS5 algorithm
|
| |
|
| Submission date |
Mar 19, 2009 |
| Last update date |
Oct 08, 2010 |
| Contact name |
Penelope Anne Morel |
| E-mail(s) |
morel@pitt.edu
|
| Phone |
412 624 0343
|
| Fax |
412 383 8098
|
| Organization name |
University of Pittsburgh
|
| Department |
Immunology
|
| Street address |
200 Lothrop Street, BST E1048
|
| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15261 |
| Country |
USA |
| |
|
| Platform ID |
GPL81 |
| Series (1) |
| GSE15320 |
Microarray analysis of therapeutic (GM4) and non therapeutic (GM) NOD DC |
|
