|
| Status |
Public on Mar 19, 2012 |
| Title |
App1_NE_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Actinobacillus pleuropneumoniae 4074
|
| Organism |
Actinobacillus pleuropneumoniae serovar 1 str. 4074 |
| Characteristics |
culture condition: cultured in TSB medium to middle exponential phase
treatment: control
|
| Treatment protocol |
For catecholamines treated A. pleuropneumoniae samples, epinephrine (Epi) and norepinephrine (NE) were added respectively into the medium at 50μM.
|
| Growth protocol |
A. pleuropneumoniae was cultured in Tryptic Soy Broth (TSB) or on Tryptic Soy Agar (TSA) (Difco Laboratories) supplemented with 10 μg/ml of nicotinamide adenine dinucleotide (NAD) and 10% (v/v) filtered cattle serum at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNA-Solv Reagent (Omega) according to the manufacturer’s instructions, then purified using QIAGEN RNeasy Mini Kit (QIAGEN).
|
| Label |
cy5
|
| Label protocol |
2μg RNA was reverse-transcribed into cDNA and then transcribed into cRNA. After purified, 4μg cRNA from control sample was labeled with Cy5 NHS ester and 4μg cRNA of Epi or NE treated sample was labeled with Cy3 NHS ester (GE healthcare) respectively and purified.
|
| |
|
| Channel 2 |
| Source name |
Norepinephrine (NE) treated Actinobacillus pleuropneumoniae
|
| Organism |
Actinobacillus pleuropneumoniae |
| Characteristics |
culture condition: cultured in TSB medium with 50 μM norepinephrine to middle exponential phase
treatment: Norepinephrine
|
| Treatment protocol |
For catecholamines treated A. pleuropneumoniae samples, epinephrine (Epi) and norepinephrine (NE) were added respectively into the medium at 50μM.
|
| Growth protocol |
A. pleuropneumoniae was cultured in Tryptic Soy Broth (TSB) or on Tryptic Soy Agar (TSA) (Difco Laboratories) supplemented with 10 μg/ml of nicotinamide adenine dinucleotide (NAD) and 10% (v/v) filtered cattle serum at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNA-Solv Reagent (Omega) according to the manufacturer’s instructions, then purified using QIAGEN RNeasy Mini Kit (QIAGEN).
|
| Label |
cy3
|
| Label protocol |
2μg RNA was reverse-transcribed into cDNA and then transcribed into cRNA. After purified, 4μg cRNA from control sample was labeled with Cy5 NHS ester and 4μg cRNA of Epi or NE treated sample was labeled with Cy3 NHS ester (GE healthcare) respectively and purified.
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed using Gene Expression Hybridization Kit (Agilent). After fragmentation, 300ng of Cy5 labeled cRNA and Cy3 labeled cRNA were hybridized with the same array at 65°C, 10 rpm rotation for 17 hours. Then, the arrays were washed twice 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 |
biological replicate 2 of 3 for norepinephrine treated Actinobacillus pleuropneumoniae
|
| Data processing |
We used and submitted only one probe signal (the probe that near the 3 end of the gene) for each gene in this study. The 2477 rows in the sample data table represented 2477 ORFs on the array. The data were analyzed using Feature Extraction Software (Agilent). The signal intensities were normalized using Feature Extraction Software (Agilent) and transformed into log2 values.
|
| |
|
| Submission date |
Nov 20, 2010 |
| Last update date |
Mar 19, 2012 |
| 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) |
| GSE25516 |
Expression data of Actinobacillus pleuropneumoniae 4074 in response to epinephrine and norepinephrine |
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