|
| Status |
Public on Jul 23, 2014 |
| Title |
Spleen-21dpi-control-26 |
| Sample type |
RNA |
| |
|
| Source name |
Spleen-21dpi-control-26
|
| Organism |
Gallus gallus |
| Characteristics |
strain: Guangxi Yellow
tissue: Spleen
age: 21 days
|
| Treatment protocol |
On the first day, the chicks in the SPF isolators were allowed to eat and drink ad libitum to stabilize their physiological status. On the morning of the second day, eighty chicks were assigned to one SPF isolator and thirty-five to the other. The eighty chicks were challenged orally with 0.5 ml bacterial culture containing 2×108 CFU of S. Pullorum, and the remaining thirty five chicks (the control group) were mock-challenged with distilled water, and this process was completed by two technicians within half an hour. After the last chick was inoculated with bacterial broth, eight challenged chicks and three control chicks were randomly selected. They were weighed, and 0.2 ml-1 ml blood was drawn for testing cytokines and antibodies via ELISA. The chicks were then sacrificed via cervical dislocation immediately after anesthesia with 150 mg/kg sodium pentobarbital. This process was repeated at 2 hours post-infection (hpi), 4 hpi, 8 hpi, 24 hpi, 3 days post-infection (dpi), 5dpi, 7dpi, 12dpi, and 21dpi.
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| Growth protocol |
For this study, one hundred and fifteen Chinese indigenous half-sibling one-day-old Guangxi Yellow chicks were obtained from the Guangdong Poultry Science Institute. All of the chicks were transported in a constant temperature incubator at 32°C. Those chicks were divided into 2 groups and were reared in two separate SPF isolators that were kept sterilized before the chicks entered. Thereafter, the SPF isolators were maintained at temperatures no less than 30°C with 24 hours of light for the first week and temperatures no less than 28°C and 12:12 (light:dark) hours thereafter. The birds were fed sterilized feed (autoclaved) and sterile still water ad libitum without any addition of antibiotics. The chicken manure was cleaned every two days to reduce the chances of re-infection. All of the instruments or tools needed were sterilized (via spraying bromogeramine diluent on the surface) before being sent to the isolators.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted by Trizol (Life Technology, 15596-018) following the manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions: (complimentary RNA) with a sample input RNA range between 10 ng and 200 ng of total RNA or a minimum of 5 ng of poly A+ RNA for one-color processing. The method uses T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3-labeled CTP. Amplification is typically at least a 100-fold from total RNA to cRNA with the use of this kit.
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| |
|
| Hybridization protocol |
Add 500 μL of nuclease-free water to the vial containing lyophilized 10X Blocking Agent supplied with the Agilent Gene Expression Hybridization Kit, or add 1250 μL of nuclease-free water to the vial containing lyophilized large volume 10X Blocking Agent (Agilent p/n 5188-5281).Mix by gently vortexing. If the pellet does not go into solution completely, heat the mix for 4 to 5 minutes at 37°C.Drive down any material adhering to the tube walls or cap by centrifuging for 5 to 10 seconds.
|
| Scan protocol |
Put assembled slide holders with or without the ozone-barrier slide cover into the scanner carousel.In the Scan Control main window, choose the slot number of the first slide for Start Slot and the slot number for the last slide for End Slot.For 4x44K microarrays, select Profile AgilentG3_GX_1Color. Verify scan settings for one-color scans. Verify that the Scanner status in the main window says Scanner Ready.In the Scan Control main window, click Scan Slot m-n where m is the slot of the first slide, and n is the slot for the last slide.
|
| Description |
Gene expression profile of spleen at 21 days post infected with Salmonella pullorum
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5 (Agilent) using default parameters (protocol G4140-90040_GeneExpression_One-color_v6.5) 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 |
Jul 22, 2014 |
| Last update date |
Jul 23, 2014 |
| Contact name |
Teng Ma |
| E-mail(s) |
[email protected]
|
| Phone |
015303040690
|
| Organization name |
Yangzhou University
|
| Department |
College of Animal Science and technology
|
| Street address |
88 South University ave.
|
| City |
Yangzhou |
| State/province |
Jiangsu |
| ZIP/Postal code |
225009 |
| Country |
China |
| |
|
| Platform ID |
GPL15357 |
| Series (1) |
| GSE59663 |
Expression profiles in different stages of the immune response to Salmonella enterica Pullorum in the chicken spleen |
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