|
| Status |
Public on Mar 29, 2013 |
| Title |
saline_PBS_1dpi_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Mouse lung, mock-treated with saline, mock-infected with PBS, 1 days post-infection
|
| Organism |
Mus musculus |
| Characteristics |
strain: BALB/c
gender: female
tissue: lung
b240 or saline treatment: saline
ca04 or pbs treatment: PBS
time: 1 days post-infection
|
| Treatment protocol |
We conducted two types of experiments to determine the effects of b240 administration on host responses in mice. In both experiments, mice were orally mock-administered with saline or administered with heat-killed Lactobacillus pentosus b240 at a dose of 10 mg/mouse daily for 21 days prior to infection and for 14 days after infection or mock-infection. (i) To define the effects of b240 administration on gene expression, mice were infected with 10 MLD50 of CA04 virus on days 21 post-b240 administration and euthanized on days 1, 3, and 6 post-infection. (ii) To investigate the immune responses induced by b240 administration in the lungs of mice, mice were mock-infected with PBS on day 21 post-b240-administration and euthanized on days -7, 0, 1, 3, and 6 post-infection. These lung tissues were immediately frozen at -80°C until processing and subjected to microarray analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The lungs of mice were homogenized with TRIzol Reagent (Invitrogen) at a final concentration of 5% w/v. The homogenized materials were transferred to a fresh 1.5 ml tube to which chloroform-isoamyl alchol (5:1) was added. The tube was vortexed and then 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 100 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 Mouse GE 8x60K Microarrays (G4852A) 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 in the lungs of saline-treated mice at 1 days post-mock-infection.
|
| 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 |
Jan 25, 2013 |
| Last update date |
Mar 29, 2013 |
| 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 |
GPL13912 |
| Series (1) |
| GSE43764 |
Protective efficacy of orally administered, heat-killed Lactobacillus pentosus b240 against influenza A virus |
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