|
| Status |
Public on Jun 08, 2018 |
| Title |
BALF_exosome_PR8_infection_48hpi_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
PR8 infection, 48hpi, BALF exosome, replicate 3
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Blonchoalveolar lavage fluid exosome
stimulant: PR8 infection at 48 hpi
|
| Treatment protocol |
Mouse lungs were lavaged with 1 ml of phosphate-buffered saline (PBS). Lavage fluid (800 µl) was collected by gentle aspiration. This procedure was repeated three times. BALF was centrifuged at 1,100 × g for 15 min at 4°C, and the cells and supernatants were collected. BALF was centrifuged at 16,000 × g for 60 min at 4°C. To remove cellular debris, the recovered supernatants were filtered through a 0.22-μm Millex-GV (Millipore, MA, USA). The supernatant was then ultracentrifuged at 100,000 × g for 90 min at 4°C. The pellets containing the exosomes were resuspended in 100 µl of PBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA from the exosomes was isolated by using the miRNeasy Serum/Plasma Kit (QIAGEN). Isolated total RNA integrity was evaluated by using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and by examining 28S/18S ribosomal RNA bands with an Agilent 2100 bioanalyzer (Agilent Technologies) according to the manufacturer’s instructions. We confirmed that the RNA isolated from the exosomes did not contain ribosomal RNAs.
|
| Label |
Cy3
|
| Label protocol |
One hundred nanograms of total RNA was used for miRNA microarray by using the Agilent miRNA Complete Labeling and Hyb Kit (Agilent Technologies) according to the manufacturer’s instructions. Cy3-pCp-labelled RNA was dried by a vacuum concentrator at 40°C for 2 h.
|
| |
|
| Hybridization protocol |
Cy3-pCp-labelled RNA was resuspended in hybridization master mix and was hybridized on the Agilent SurePrint G3 Mouse miRNA 8 × 60 K V19 at 55°C for 20 h.
|
| Scan protocol |
The microarray slides were scanned using an Agilent High-Resolution Microarray Scanner (Agilent Technologies), and image data were processed by using Agilent Feature Extraction software ver. 10.7.3.1.
|
| Description |
miRNA expression in BALF exosome in PR8 infection at 48 hpi
|
| Data processing |
All data were subsequently uploaded into GeneSpring GX ver. 13.0 for data analysis. We selected miRNAs that were filtered on Expression (20–100th) percentile in the Normalized data and were flagged as Detected. Then, we selected miRNAs whose expression was statistically significantly different by moderated t-test (naive vs. 48 hpi, and naive vs. 72 hpi) and 2-fold higher than those in the naive mice group.
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| |
|
| Submission date |
Nov 17, 2017 |
| Last update date |
Jun 09, 2018 |
| Contact name |
Tadashi Maemura |
| E-mail(s) |
[email protected]
|
| Organization name |
Influenza Research Institute
|
| Street address |
575 Science Drive
|
| City |
Madison |
| State/province |
Wisconsin |
| ZIP/Postal code |
53705 |
| Country |
USA |
| |
|
| Platform ID |
GPL17912 |
| Series (1) |
| GSE107107 |
Exosomal microRNA (miRNA) expression in Blonchoalveolar lavage fluid (BALF) collected from mice infected with influenza virus and inoculated with poly(I:C). |
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