|
| Status |
Public on Jun 30, 2014 |
| Title |
App1_Ana_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Actinobacillus pleuropneumoniae 4074
|
| Organism |
Actinobacillus pleuropneumoniae serovar 1 str. 4074 |
| Characteristics |
strain: reference strain of serovar 1
culture condition: cultured in TSB medium aerobically
|
| Treatment protocol |
The bacteria was cultured under aerobic condition to mid-log phase (3 hours) and then divided into two separate groups, one group was continually cultured under aerobic condition for 1 hour and the other group was cultured under anaerobic condition for 1 hour.
|
| 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 (aerobically cultured) was labeled with Cy5 NHS ester and 4μg cRNA of test sample (anaerobically cultured) was labeled with Cy3 NHS ester (GE healthcare) respectively and purified.
|
| |
|
| Channel 2 |
| Source name |
Actinobacillus pleuropneumoniae 4074
|
| Organism |
Actinobacillus pleuropneumoniae serovar 1 str. 4074 |
| Characteristics |
strain: reference strain of serovar 1
culture condition: cultured in TSB medium anaerobically
|
| Treatment protocol |
The bacteria was cultured under aerobic condition to mid-log phase (3 hours) and then divided into two separate groups, one group was continually cultured under aerobic condition for 1 hour and the other group was cultured under anaerobic condition for 1 hour.
|
| 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 (aerobically cultured) was labeled with Cy5 NHS ester and 4μg cRNA of test sample (anaerobically cultured) 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 comparing gene expressions of Actinobacillus pleuropneumoniae cultured aerobically and anaerobically
|
| Data processing |
The scanned signal intensities were normalized using Feature Extraction Software (Agilent) by Linear & LOWESS method (the default method for normalizing Agilent microarrays). Then, the normalized signal intensities were transformed into log2 values. The data submitted are normalized log2 ratio (Cy5/Cy3) representing test/reference.
For further analysis, the genes with positive signals (flags = P or M) in all the three replicates were selected. The genes with P value < 0.05 (one-way class T-test) using the software TM4 were selected as differentially expressed genes to do function analysis and experimental validations.
|
| |
|
| Submission date |
Aug 01, 2012 |
| Last update date |
Jun 30, 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 |
GPL9691 |
| Series (1) |
| GSE39801 |
Expression data of Actinobacillus pleuropneumoniae 4074 under aerobic and anaerobic environment |
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