|
| Status |
Public on Oct 11, 2011 |
| Title |
Caco-2-TLR4-D299G, rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Caco-2 cells stably transfected with TLR4-D299G
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Caco-2
cell type: intestinal epithelial
|
| Treatment protocol |
Prior to analysis, cells (1.5 x 105 / ml) of Caco-2 stable cell lines expressing TLR4-WT, TLR4-D299G, TLR4-T399I or untransfected were cultured for 8 days in all experiments.
|
| Growth protocol |
HA-tagged TLR4-wildtype plasmid (TLR4-WT) was obtained from InvivoGen. TLR4 mutants (D299G, T399I) were generated within the TLR4-WT expression construct and confirmed by sequencing (Trenzyme). Plasmids (EndoFree Maxi, Qiagen) were transfected into Caco-2 cells on a 12-well-plate (poly-D-lysine coated) using 1µg/well of TLR4-WT, TLR4-D299G or TLR4-T399I (Lipofectamine LTX, Invitrogen). Stable IEC transfectant clones expressing TLR4-WT, TLR4-D299G or TLR4-T399I were selected with 1 µg/ml blasticidin (InvivoGen). Controls were untransfected cells without any exogenous DNA. Transfection efficiency was evaluated by semiquantative RT-PCR analysis, as well as western blotting and immunohistochemistry with anti-HA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell samples were frozen in trizole at -80°C until further processing. Total RNA was isolated (RiboPure, Ambion) and purified (Rneasy, Qiagen) according to the manufacturers' recommendations. All RNA samples (n=3 per condition) were analyzed independently and microarray processing was performed at The Microarray Facility (Tübingen), according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Samples were enzymatically amplified, fragmented and biotinylated using GeneChip® Whole Transcript (WT) Sense Target Labeling Kit (Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 15µg of cDNA were hybridized for 16h at 45°C on GeneChip HuGene 1.0 ST v1 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450 with fluidics protocol FS450_0001.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GCS3000 gene chip scanner using GCOS version 1.4 software with default settings.
|
| Description |
Gene expression data from Caco-2 cells stably transfected with TLR4-D299G
|
| Data processing |
Raw CEL-files were imported into Expression Console 1.0 (Affymetrix). RMA was used for array normalization and signal calculation.
|
| |
|
| Submission date |
Dec 21, 2010 |
| Last update date |
Oct 11, 2011 |
| Contact name |
Elke Cario |
| E-mail(s) |
elke.cario@uni-due.de
|
| Phone |
+49-201-723-4527
|
| Organization name |
University of Duisburg-Essen, University Hospital of Essen
|
| Department |
Division of Gastroenterology and Hepatology
|
| Lab |
Cario
|
| Street address |
Virchowstr. 171
|
| City |
Essen |
| State/province |
NRW |
| ZIP/Postal code |
45147 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE26226 |
Expression data from Caco-2 cells expressing TLR4 and associated mutants |
|
