|
| Status |
Public on Mar 31, 2010 |
| Title |
serotype 10 reference strain |
| Sample type |
RNA |
| |
|
| Source name |
serotype 10 strain D13039
|
| Organism |
Actinobacillus pleuropneumoniae |
| Characteristics |
strain: D13039
|
| 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 mid-log phase and the total RNA were extracted using Trizol reagent according to the manufacturer’s instructions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells using Trizol reagent (Invitrogen) according to the manufacturer’s protocol and the RNA concentration was measured on a NanoDrop ND-1000 UV spectrometry. The treated RNA was purified with a 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 stirred with DMSO and 0.3M NaHCO3 and then Cy3 NHS ester (GE healthcare) were added. 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 |
Scanned on an Agilent G2565BA scanner.
|
| Description |
cultured in TSB medium to middle exponential phase
|
| Data processing |
Images were quantified using Agilent Feature Extraction Software (version 9.5). The values of signal intensity were normalized and transformed into log base 2.
|
| |
|
| Submission date |
Apr 03, 2009 |
| Last update date |
Sep 03, 2014 |
| 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 |
GPL8394 |
| Series (1) |
| GSE15545 |
Gene expression profiling of distinct serotypes of Actinobacillus pleuropneumoniae |
|
