|
| Status |
Public on Sep 09, 2010 |
| Title |
LB vs low iron array #1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
LB
|
| Organism |
Proteus mirabilis HI4320 |
| Characteristics |
strain: HI4320
medium: LB broth
|
| Growth protocol |
Cultured in LB broth at 37C to OD600 = 0.744 (mid-log).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1 ml of culture was added to 2 ml RNA Protect. RNA was isolated using the RNEasy kit (Qiagen) according to the manufacturer’s instructions, except that 3 mg/ml lysozyme was added to cells for 15 min. DNA was digested using Turbo-DNAfree DNase (Ambion).
|
| Label |
Cy5
|
| Label protocol |
RNA (4 μg) was used as a template for cDNA synthesis and was labeled with Cyanine 5 (GE Healthcare) according to SOP#M007 from The Institute for Genomic Research (TIGR).
|
| |
|
| Channel 2 |
| Source name |
low iron
|
| Organism |
Proteus mirabilis HI4320 |
| Characteristics |
strain: HI4320
medium: LB broth + Desferal
|
| Growth protocol |
Cultured in LB broth + 15uM Desferal at 37C to OD600 = 0.604 (mid-log).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1 ml of culture was added to 2 ml RNA Protect. RNA was isolated using the RNEasy kit (Qiagen) according to the manufacturer’s instructions, except that 3 mg/ml lysozyme was added to cells for 15 min. DNA was digested using Turbo-DNAfree DNase (Ambion).
|
| Label |
Cy3
|
| Label protocol |
RNA (4 μg) was used as a template for cDNA synthesis and was labeled with Cyanine 3 (GE Healthcare) according to SOP#M007 from The Institute for Genomic Research (TIGR).
|
| |
|
| |
| Hybridization protocol |
Labeled cDNA was mixed and hybridized to an array slide using TIGR SOP#M008 (ftp://ftp.jcvi.org/pub/data/PFGRC/pdf_files/protocols/M008.pdf).
|
| Scan protocol |
The microarray was scanned using a ScanArray Express microarray scanner (Perkin Elmer) at 10 μm resolution and quantified using the ScanArray Express software.
|
| Description |
This array compared the P. mirabilis transcriptome during logarithmic-phase growth in LB broth to logarithmic-phase growth in iron-restricted LB broth. This is array #1 of 5.
|
| Data processing |
The LOWESS normalization algorithm (ScanArray Express, Perkin Elmer) was used to normalize microarray data. Median spot values for each triplicate probe were extracted using MIDAS (TIGR). A gene was considered differentially regulated if it was greater than 2-fold changed in LB compared to LB + Desferal.
|
| |
|
| Submission date |
Sep 09, 2009 |
| Last update date |
Sep 10, 2009 |
| Contact name |
Melanie M Pearson |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Michigan Medical School
|
| Department |
Microbiology and Immunology
|
| Lab |
Melanie Pearson
|
| Street address |
1150 W Medical Center Dr
|
| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109-0620 |
| Country |
USA |
| |
|
| Platform ID |
GPL9078 |
| Series (1) |
| GSE18051 |
Functional redundancy of iron acquisition systems is required for Proteus mirabilis pathogenesis during UTI |
|
