|
| Status |
Public on Apr 20, 2007 |
| Title |
P.stutzeri A1501 treated with 20mM ammonia and 0.5% Oxygen tension-1 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Genomic DNA of P. stutzeri A1501
|
| Organism |
Stutzerimonas stutzeri A1501 |
| Characteristics |
strain: A1501
|
| Treatment protocol |
the total DNA of the P.stutzeri A1501 without any treatment.
|
| 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.
|
| |
|
| 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 20mM ammonia and 0.5% Oxygen tension. The bacterium was collected simultaneous with the bacterium treated with 0.1mM ammonia and 0.5% Oxygen tension when the nitrogenase activity was detectable.Then the bacterium was collected and extracted the total RNA.
|
| 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 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 |
P.stutzeri A1501 treated with 20mM ammonia and 0.5% Oxygen tension-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.
|
| |
|
| Submission date |
Dec 19, 2006 |
| 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) |
| GSE6572 |
Microarray was used to study the gene expression of P. stutzeri A1501 under different growth conditions |
|
| Data table header descriptions |
| ID_REF |
|
| log_ratio-not_normalized |
|
| 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) |
