|
| Status |
Public on Feb 10, 2010 |
| Title |
Expression profiling of P. stutzeri A1501 treated with 20mM ammonia shock for 10 min-1 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Genomic DNA of P. stutzeri A1501
|
| Organism |
Stutzerimonas stutzeri A1501 |
| Characteristics |
strain: A1501
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The genomic DNA was extracted as the Protocol from the TIANamp Bacteria DNA kit from TIANGEN BIOTECH (BEIJING) CO., LTD
|
| Label |
cy3
|
| Label protocol |
The genomic DNA were then fluorescently labeled using BioPrime DNA Labeling System (Life Technologies/Invitrogen; Carlsbad, CA) following the manufacturer’s instructions. The genomic DNA was added to 2.5× random primers solution (50 mM Tris-HCl, pH6.8; 5 mM MgCl2; 10 mM 2-mercaptoethanol; 300 μg/ml oligodeoxyribonucleotide primer), boiled at 100 °C for 5 min and then chilled on ice. 10× dNTP Mix for DNA labelling (0.12 mM dATP, dGTP,dTTP; 0.06 mM dCTP; 1 mM Tris-HCl, pH 8.0; 0.1 mM EDTA); Cy3-dCTP (0.06 mM); Klenow fragment (40 U) were added. The mixture was briefly centrifugated and was incubated at 37°C overnight away from light. 5μl 0.5 M EDTA (pH 8.0) was used to stop reaction.
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| |
|
| Channel 2 |
| Source name |
The cDNA of P. stutzeri A1501 - treated
|
| Organism |
Stutzerimonas stutzeri A1501 |
| Characteristics |
strain: A1501
|
| Treatment protocol |
The bacterium was treated with 0.1 mM ammonia and 0.5% Oxygen tension until the nitrogenase activity was detectable. Then the cells were sudden shifted from the nitrogen fixation conditions to the ammonia repression conditions by addition of 20 mM ammonia for 10min. Subsequently, the bacterium was collected and began the RNA extraction process.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA was extracted as the Protocol from the SV Total RNA Isolation System (Promega)
|
| Label |
cy5
|
| Label protocol |
The cDNA were then fluorescently labeled using BioPrime DNA Labeling System (Life Technologies/Invitrogen; Carlsbad, CA) following the manufacturer’s instructions. The cDNA was added to 2.5× random primers solution (50 mM Tris-HCl, pH6.8; 5 mM MgCl2; 10 mM 2-mercaptoethanol; 300 μg/ml oligodeoxyribonucleotide primer), boiled at 100 °C for 5 min and then chilled on ice. 10× dNTP Mix for DNA labelling (0.12 mM dATP, dGTP,dTTP; 0.06 mM dCTP; 1 mM Tris-HCl, pH 8.0; 0.1 mM EDTA); Cy5-dCTP (0.06 mM); Klenow fragment (40 U) were added. The mixture was briefly centrifugated and was incubated at 37°C overnight away from light. 5μl 0.5 M EDTA (pH 8.0) was used to stop reaction.
|
| |
|
| |
| Hybridization protocol |
Labeled cDNA/DNA was purified using QIAquick columns and mixed with 50× Denharts; 20× SSC; yeast tRNA 24 μg; 1 mM HEPES, 10% SDS, the mixture was heated at 100 °C for 2 min and cooled to room temperature and applied to the array slides under glass coverslips. Hybridization was performed at 65°C overnight in a Micro hybridization incubator (Robbins Scientific; Sunnyvale, CA).
|
| Scan protocol |
GenePix 6.0 software (Axon Instruments, Inc.) was used for image analysis and data visualization.
|
| Description |
Expression profiling of P. stutzeri A1501 treated with 20mM ammonia shock for 10 min-1
|
| Data processing |
First, the signals were normalized using with the GenePix 6.0 software and then with a locally weighted scatterplot smoothing regression (LOWESS) algorithm in the MIDAS software package.
Genes were considered to be differentially expressed if (i) average expression changed by at least 2-fold in three independent experiments performed with triplicate RNA samples or (ii) the change in gene expression was in the same direction (“increased” or “decreased”) in three experiments.
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| |
|
| Submission date |
Feb 10, 2009 |
| Last update date |
Feb 11, 2009 |
| Contact name |
yongliang yan |
| E-mail(s) |
yongliangyan@yahoo.com.cn
|
| Organization name |
CAAS
|
| Street address |
zhongguancun south 12th
|
| City |
Beijing |
| ZIP/Postal code |
100081 |
| Country |
China |
| |
|
| Platform ID |
GPL4677 |
| Series (1) |
| GSE14775 |
Expression profiling of P. stutzeri A1501 treated with 20mM ammonia shock for 10 min |
|
| Data table header descriptions |
| ID_REF |
|
| log_ratio-not_normalized |
Not normalized, log2 ratio of (635/532) |
| F635 Median |
Channel 1 median Intensity (Foreground) |
| F635 SD |
Standard deviation of Channel 1 median Intensity (Foreground) |
| B635 Median |
Channel 1 median Intensity (Background) |
| B635 SD |
Standard deviation of Channel 1 median Intensity (Background) |
| F532 Median |
Channel 2 median Intensity (Foreground) |
| F532 SD |
Standard deviation of Channel 2 median Intensity (Foreground) |
| B532 Median |
Channel 2 median Intensity (Background) |
| B532 SD |
Standard deviation of Channel 2 median Intensity (Background) |
| VALUE |
normalized log2 ratio (Cy5/Cy3) |
