|
| Status |
Public on Jul 01, 2014 |
| Title |
WP3Δhns-20C rep6 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
WP3 cells at mid-exponential phase
|
| Organism |
Shewanella piezotolerans WP3 |
| Characteristics |
cell type: WP3
growth protocol: cultured at 20C in 2216E medium
genotype/variation: WP3Δhns
|
| Growth protocol |
Shewanella strains were cultured in modified marine medium 2216E (5 g/l tryptone, 1 g/l yeast extract, 0.1 g/l FePO4, 34 g/l NaCl) shaking at 220 rpm at 20°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
The retro-transcription of the 20 μg total RNA was accorded with the Invitrogen manufacturer’s instruction. cDNA labeled with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction.
|
| |
|
| Channel 2 |
| Source name |
WP3 cells at mid-exponential phase
|
| Organism |
Shewanella piezotolerans WP3 |
| Characteristics |
cell type: WP3
growth protocol: cultured at 20C in 2216E medium
genotype/variation: WP3 wild-type
|
| Growth protocol |
Shewanella strains were cultured in modified marine medium 2216E (5 g/l tryptone, 1 g/l yeast extract, 0.1 g/l FePO4, 34 g/l NaCl) shaking at 220 rpm at 20°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
The retro-transcription of the 20 μg total RNA was accorded with the Invitrogen manufacturer’s instruction. cDNA labeled with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction.
|
| |
|
| |
| Hybridization protocol |
Klenow enzyme labeling strategy was adopted after reverse transcription. Briefly, 20 μg amplified RNA was mixed with 4 μl random hexamers, denatured at 70°C for 5 min, and cooled on ice. Then, 4 μl of first-strand buffer, 2 μl of 0.1M DTT, 1 μl 10mM dNTP, and 1.5 μl SuperScipt Ⅱ (Invitrogen) were added. The mixtures were incubated for 10 min at 25°C, then for 90 min at 42°C. The cDNA products were purified using the PCR purification kit and vacuum evaporated to 10 μl, and then it was mixed with 4 μg random nanomer, heated to 95°C for 3 min, and snap cooled on ice. After that, 10 μl buffer, dNTP, and Cy5-dCTP or Cy3-dCTP (Amersham Biosciences) were added to final concentrations of 120 mM dATP, dGTP, dTTP and 60 mM dCTP, and 40mM Cy-dye. Klenow enzyme (Takara, Japan) was then added, and the reaction was performed for 90 min at 37°C. Labeled cDNA was purified with the PCR purification kit and resuspended in elution buffer.
|
| Scan protocol |
All microarrays were scanned with a LuxScan 10K canner using microarray scanner 2.3 software (CapitalBio, Beijing, China). We quantified signal intensites of individual spot from the 24-bit TIFF images using SpotData Pro 2.2 (CapitalBio, Beijing, China).
|
| Description |
raw data file: 8210 WP3cy5-hnscy3.LSR
WP3Δhns cDNA was labled with Cy3 and WP3 wild-type cDNA was labled with Cy5
|
| Data processing |
S-plus software was used for the normalization of microarray data. A spatial and intensity-dependent normalization based on a LOWESS program were used. Normalized data was log transformed and loaded into MAANOVA under R environment for multiple testing, by fitting a mixed effects ANOVA model. Normalized log2 ratio (Cy5/Cy3) representing test/reference. In all of the experiments, WP3hns is the test sample and WP3 wild-type is the reference sample. Microarray spots with P values <0.001 in the F-test and log2 ratio were regarded as differentially expressed genes. In addition, all of the DEGs were confirmed with Significance Analysis of Microarrays (SAM) software.
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| |
|
| Submission date |
May 22, 2014 |
| Last update date |
Jul 01, 2014 |
| Contact name |
Huahua Jian |
| E-mail(s) |
[email protected]
|
| Organization name |
Shanghai Jiao Tong University
|
| Street address |
No.800 Dongchuan Road
|
| City |
Shanghai |
| ZIP/Postal code |
200240 |
| Country |
China |
| |
|
| Platform ID |
GPL16568 |
| Series (2) |
| GSE57905 |
hns mutant vs Shewanella piezotolerans WP3 wild-type strain 20C |
| GSE57940 |
hns mutant vs Shewanella piezotolerans WP3 wild-type strain 4C and 20C |
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