|
| Status |
Public on Jan 13, 2010 |
| Title |
Time series time 10 minute after iron repletion, Replicate 1 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
total RNA extracted from MR-1 cells treated with ferrous sulfate for 10 minute
|
| Organism |
Shewanella oneidensis MR-1 |
| Characteristics |
strain: MR-1
agent: ferrous sulfate
time: 10 minutes
strain: MR-1
|
| Growth protocol |
MR-1 were grown in 100 ml LB medium in 500 ml shake flasks with vigorous shaking at 250–300 rpm
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNAs from MR-1 were isolated using the TRIzol Reagent (Gibco BRL) following manufacturer's instructions. After precipitation, RNA samples were purified with the Qiagen RNeasy Mini kit (Qiagen) following the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
Fluorescently Cy5-labeled cDNA copies of total cellular RNA extracted from MR-1 were prepared by incorporation of fluorescein-labeled nucleotide analogs during a first-strand reverse transcription reaction following the manufacturer's instructions (Invitrogen).
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| |
|
| Channel 2 |
| Source name |
total genomic DNA extracted from MR-1 cells grown to stationary phase (reference)
|
| Organism |
Shewanella oneidensis MR-1 |
| Characteristics |
strain: MR-1
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
MR-1 cells were lysed by repeated boiling-frozen cycles. DNA were precipitated by isoproposal after succssive extraction with phenol, phenol-chloroform and chloroform
|
| Label |
Cy3
|
| Label protocol |
MR-1 genomic DNA (gDNA) was amplified by Klenow following the manufacturer's instructions (Invitrogen), and Cy3 dUTP (Amersham Biosciences) was incorporated into the product. Fluorescein-labeled probes were purified using the Qiaquick PCR purification kit (Qiagen).
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| |
|
| |
| Hybridization protocol |
The labeled cDNA and gDNA pools were mixed and hybridized simultaneously to the array on UltraGAPS slides (Corning) in a solution containing 5x SSC, 0.1% SDS, and 0.1 mg/mL of herring sperm DNA (Gibco BRL). The entire process of hybridization, including prehybridization, hybridization, and post-hybridization wash, was performed following the manufacturer's instructions.
|
| Scan protocol |
Microarrays were scanned using the confocal laser microscope of the ScanArray Gx Microarray Analysis System (PerkinElmer) at a resolution of 5 µm per pixel. Scanning parameters (laser power and photomultiplier tube or PMT gain) were adjusted, so that overall intensities in both fluorescence channels were relatively equal and few spots were saturated.
|
| Description |
Biological replicate 1 from MR-1 sample after iron repletion by treatment with ferrous sulfate for 10 minute
|
| Data processing |
To determine fluorescence intensity (pixel density) and background intensity, 16-bit TIFF scanned images were analyzed using the software ImaGene version 7.0 (Biodiscovery). The text files from ImaGene were then analyzed using standard procedure as published in Appl Environ Microbiol 2006, 72(8):5578-5588. Specifically, The data files were subjected to Lowess intensity-based normalization and were analyzed further using GeneSpring, version 5.1 (Silicon Genetics, Redwood City, CA). To assess the statistical significance of individual data points, A statistical model incorporating per-gene variance (z values) was used to compute the posterior probability that the expression level of each gene changed in the direction indicated by the mean value. Ratios with |log3(ratio)| > 1 or |z| > 2.0 are considered significant.
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| |
|
| Submission date |
Jan 13, 2010 |
| Last update date |
Jan 13, 2010 |
| Contact name |
Yunfeng Yang |
| E-mail(s) |
yangy@ornl.gov
|
| Phone |
865-576-3998
|
| Fax |
865-576-8646
|
| Organization name |
Oak Ridge National Lab
|
| Department |
Biosciences Division
|
| Street address |
1 Bethel Valley Road
|
| City |
Oak Ridge |
| ZIP/Postal code |
37830 |
| Country |
USA |
| |
|
| Platform ID |
GPL3253 |
| Series (1) |
| GSE15334 |
MR-1 gene expression profiles of iron response |
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