|
| Status |
Public on Sep 30, 2012 |
| Title |
Mock_6h_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Monocyte-derived macrophages, not infected, 6hpi
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: monocyte-derived macrophages
treatment: mock
time post-infection: 6 hrs
|
| Treatment protocol |
Human monocyte-derived macrophages were mock-infected, or infected with IDN3006, VN3028IIcl2, or IDN3006/cl2PA at an MOI of 2. The virus inoculum was removed, and the cells were washed three times and then incubated in serum-free RPMI 1640 medium supplemented with 0.3% BSA.
|
| Growth protocol |
Peripheral blood mononuclear cells were independently separated from the buffy coat of healthy donors. Monocytes were purified by adherence and were allowed to differentiate for 14 days in RPMI 1640 medium supplemented with 5 ng/ml of recombinant human GM-CSF and 10% human serum derived from the corresponding blood donors. Cells were incubated at 37 °C in 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At 6 hpi, the cells were lysed with TRIzol Reagent (Invitrogen) and chloroform-isoamyl alchol (5:1) was added. The cells were then vortexed and centrifuged for 15 min at 12,000 g at 4 °C. The resulting aqueous layer was subjected to RNA extraction by using RNeasy Mini Kit columns (Qiagen) according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Cy3-labeled cRNA probe synthesis was initiated with 50 ng of total RNAs by using the Agilent Low Input Quick Amp Labeling kit, one color (Agilent Technologies).
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent GE hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE 8x60K Microarrays (G4851) 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 with an Agilent's High-Resolution Microarray Scanner using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green).
|
| Description |
Gene expression after 6hr in mock-infected human macrophages.
Replicate 1 of 3.
|
| Data processing |
The scanned images were analyzed with Agilent Feature Extraction Software ver. 10.7.3.1. using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Sep 09, 2012 |
| Last update date |
Sep 30, 2012 |
| Contact name |
Ryo Takano |
| E-mail(s) |
ewigkeitryo@gmail.com
|
| Phone |
+81-155-49-5645
|
| Organization name |
Obihiro University of Agriculture and Veterinary Medicine
|
| Department |
National Research Center for Protozoan Diseases
|
| Lab |
Research Unit for Global Infection Control
|
| Street address |
Inada-cho
|
| City |
Obihiro |
| State/province |
Hokkaido |
| ZIP/Postal code |
080-8555 |
| Country |
Japan |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE40711 |
Differences in cytokine production in human macrophages and in virulence in mice are attributable to the PA protein of H5N1 influenza viruses |
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