|
| Status |
Public on Aug 01, 2017 |
| Title |
mouse alveolar macrophage_aged_viralinfection_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
alveolar macrophage, from 22-24 month-old, influenza virus
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57B/6
young or aged: aged
treatment: influenza virus
|
| Treatment protocol |
C57B/6 mice of either 2-4 months-old or 22-24 months old were obtained from the National Institute of Aging rodent facility. Mice were infected with purified human influenza virus, A/Puerto Rico/8/34 (H1N1) (PR8) (Advanced Biotechnologies Inc.; MD, USA), by instillation of with 50μl PBS containing indicated dose of PR8 virus or 50μl PBS control intranasally. At day 3 post infection, the lungs were harvested and single-cell suspension was obtained by mechanical and enzymatic digestion. . After staining with fluorescent antibodies, Alveolar macrophages were sorted according to their forward- and side-scatter profiles and by their phenotype as follow: CD45+SiglecF+CD11chiF4/80+.
|
| Growth protocol |
NA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the miRNeasy micro kit (Qiagen, Valencia CA) by utilizing QIAcube (Qiagen, Valencia CA). RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent Tapestation 2200 Bioanalyzer with High Sensitivity RNA ScreenTape (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.25 ug RNA using the One-Color Low RNA Quick Amp labeling 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 |
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 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Mouse Gene Expression v2 8x60k microarray slides (G4852B) 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).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 12.0.0.7 (Agilent) using default parameters (protocol GE1_1200_Jun14 and Grid: 074809_D_F_20150624) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Jul 27, 2016 |
| Last update date |
Aug 01, 2017 |
| Contact name |
Koji Sakamoto |
| E-mail(s) |
koji.sakamoto@yale.edu
|
| Organization name |
Yale University
|
| Department |
Internal Medicine
|
| Lab |
Naftali Kaminski
|
| Street address |
300 Cedar Street
|
| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06521 |
| Country |
USA |
| |
|
| Platform ID |
GPL21163 |
| Series (1) |
| GSE84901 |
Gene expression in murine alveolar macrophage: effect of aging and influenza infection |
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