|
| Status |
Public on Nov 09, 2011 |
| Title |
APP-1_time3_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Reference strain of serovar 1, WT
|
| Organism |
Actinobacillus pleuropneumoniae |
| Characteristics |
strain: 4074
phase: late exponential
|
| Growth protocol |
A. pleuropneumoniae strains were cultured in TSB medium supplemented with 10 μg/ml of nicotinamide adenine dinucleotide (NAD) and 10% (v/v) filtered cattle serum at 37°C.The samples were collected from early exponential phase, middle exponential phase, late exponential phase and stationary phase respectively and the total RNA were extracted using Trizol reagent according to the manufacturer’s instructions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA were extracted using RNA-Solv Reagent (Omega) according to the manufacturer’s instructions. Total RNA were purified using QIAGEN RNeasy Mini Kit (QIAGEN).
|
| Label |
cy3
|
| Label protocol |
cDNA were transcribed into cRNA using T7 RNA Polymerase and aaUTP (Ambion). cRNA were purified using QIAGEN RNeasy Mini kit (QIAGEN). 4μg cRNA were combined with DMSO and 0.3M NaHCO3 and then were added into Cy3 NHS ester (GE healthcare). The mixture were incubated at 25°C for 1 hour. 4M Hydroxylamine were added and incubated at 25°C for 15min. The labeled cRNA were purified using QIAGEN RNeasy Mini kit (QIAGEN).
|
| |
|
| Hybridization protocol |
Hybridization was performed using Gene Expression Hybridization Kit (Agilent). 1μg of cRNA were mixed with blocking agent, fragmentation buffer and incubated at 60°C for 30 min for fragmentation. GEx Hybridization Buffer were added and 100 μl mixture were hybridized at 65°C for 17 hours with 10 rpm rotation. The arrays were washed two times using wash buffer 1 and 2 from Gene Expression Wash Buffer Kit (Agilent).
|
| Scan protocol |
Arrays were scanned using Agilent Microarray Scanner System G2565BA (Agilent) with resolution of 5μm. The scanner uses 100% and 10% PTM respectively and combines the two data automatically.
|
| Description |
Two biological repetitions were combined
|
| Data processing |
The data were analyzed using Feature Extraction Software (Agilent). The signal intensity were normalized and transformed into log2 values.
|
| |
|
| Submission date |
Nov 17, 2009 |
| Last update date |
Nov 10, 2011 |
| Contact name |
Lu Li |
| E-mail(s) |
sakura.tree@163.com, xuzf@mail.hzau.edu.cn, xuzhuofei@sohu.com, kobe2071@tom.com
|
| Organization name |
Huazhong Agricultural University
|
| Street address |
Shizishan Street 1
|
| City |
Wuhan |
| State/province |
Hubei |
| ZIP/Postal code |
430070 |
| Country |
China |
| |
|
| Platform ID |
GPL9691 |
| Series (1) |
| GSE19056 |
Expression data of Actinobacillus pleuropneumoniae 4074 and the ΔluxS mutant of Actinobacillus pleuropneumoniae 4074 |
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