|
| Status |
Public on Nov 04, 2019 |
| Title |
ExVivoBlood_P150_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral Blood,infected with M.bovis P150
|
| Organism |
Bos taurus |
| Characteristics |
tissue: whole blood
cell type: PBMC
gender: male
age: < 1.5 month
infection: M.bovis P150
|
| Treatment protocol |
Each calf was nasal inoculated with 10^9 CFU of HB0801 strain, or P150 strain respectively, in 5 mL of PPLO broth. The NC calve were mock inoculated with an equal volume of sterile PPLO broth. After 7 days post-incubated, PBMCs were isolated from blood samples on a Ficoll gradient, washed with phosphate-buffered saline (PBS), and suspended in RPMI-1640 complete medium with 10% FBS with volumes equal to original blood samples. Then the PBMCs were inoculated in a 12-well plate at a density of 2-4 × 10^6 live cells each well, and co-incubated with 2μg for each well heat-killed either HB0801 or P150 M. bovis strains. The PBMCs from the NC group were added with correspond volume of sterile cell culture medium. After 72 h, the cells were harvested and disposed for microarray assay.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Lyse cells were homogenized in TRIzol Reagent by repetitive pipetting. RNA was prepared with phase separation with chloroform, precipitation with isopropyl alcohol, and washed with 75% ethanol and last redissolved. Total RNA clean-up was performed with RNasey Mini Kit(Qiagen p/n 74104), and 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 0.5 ug RNA using the One-Color Quick Amp Labeling Kit (Agilent p/n 5190-0442) according to the manufacturer's instructions, followed by RNeasy Mini Kit (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 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 Microarray Hybridization Chambers (Agilent p/n G2530-60029) 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 Microarray Scanner (Agilent p/n G2565BA) 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 7days in M.bovis P150-infected calf's blood
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Feb 13, 2018 |
| Last update date |
Nov 04, 2019 |
| Contact name |
JIN CHAO |
| E-mail(s) |
[email protected]
|
| Organization name |
Huazhong Agricultural University
|
| Street address |
1#, Shizishan Street
|
| City |
Wuhan |
| State/province |
Hubei |
| ZIP/Postal code |
430070 |
| Country |
China |
| |
|
| Platform ID |
GPL11649 |
| Series (1) |
| GSE110517 |
Transcriptome profiles of the different expression genes in peripheral blood mononuclear cells (PBMCs) of calves infected with two different virulent Mycoplasma bovis strains |
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