|
| Status |
Public on Jan 19, 2010 |
| Title |
BMDM control 3 6h |
| Sample type |
RNA |
| |
|
| Source name |
BMDM, 6h, control, replicate 3
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Bone-marrow derived macrophages
|
| Treatment protocol |
BMDM were infected with T. whipplei (MOI 50) or left untreated in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. The cultures were incubated for 6 at 37 ºC in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the RNeasy kit (QIAGEN, Valencia, CA) following the manufacturer's recommendations. The protocol includes an on-column DNase digestion. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 300 ng RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays (G4122F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after 6 hours incubation of uninfected BMDM
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5.1.1 (Agilent) using default parameters (protocol GE1-v5_95_Feb07 and Grid: 014868_D_20060807mouse) to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
May 20, 2009 |
| Last update date |
Feb 09, 2010 |
| Contact name |
Benoit Desnues |
| E-mail(s) |
benoit.desnues@univmed.fr
|
| Organization name |
Faculté de Médecine de la Timone
|
| Department |
URMITE UMR CNRS 6236
|
| Street address |
27 boulevard Jean Moulin
|
| City |
Marseille |
| ZIP/Postal code |
13385 cedex 05 |
| Country |
France |
| |
|
| Platform ID |
GPL4134 |
| Series (3) |
| GSE16180 |
Bone-marrow derived macrophages response to the Whipple's disease agent, Tropheryma whipplei |
| GSE20207 |
Wild-type bone-marrow derived macrophages response to Escherichia coli lipopolysaccharide (LPS) |
| GSE20210 |
Role of interleukin-16 in macrophage responses to Tropheryma whipplei and lipopolysaccharide |
|
