|
| Status |
Public on Jul 25, 2008 |
| Title |
E. coli NaCl hyperosmotic treatment (biological replicate 1) |
| Sample type |
RNA |
| |
|
| Source name |
E. coli Frag1, hyperosmotic NaCl treatment (biological replicate 1)
|
| Organism |
Escherichia coli |
| Characteristics |
E. coli, strain Frag1
|
| Biomaterial provider |
TIAR, School of Agricultural Science, University of Tasmania
|
| Treatment protocol |
Cell suspension was ammended with 1.37 M NaCl to obtain an osmotic pressure of 2.7 Os kg-1 for 10 minutes. The biomass was centrifuged at 4000 rpm and the pellet treated with RNAProtect (Qiagen) following the manufacturer's protocol.
|
| Growth protocol |
Grown in Davis's Mineral Salts medium (Gibco BRL Life Technologies Inc.) with 0.1% D-glucose at 25 C (100 r.p.m. shaking) until an OD (550 nm) of 0.6.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was obtained using the RNAeasy MIDI RNA extraction kit (Qiagen) using lysis buffer containing 0.1% beta-mercaptoethanol. RNA was extracted from lysates and purified according to the manufacturer’s standard protocol and subsequently stored at -80°C. RNA quality was assessed with electrophoresis using a RNA bioanalyser 2100 and Lab RNAChip (Agilent Technologies) before progression to microarray hybridization.
|
| Label |
biotin
|
| Label protocol |
Gene expression was assessed on Affymetrix E. coli Genome GeneChip arrays (Affymetrix, Inc., Santa Clara, Calif.). Briefly, total RNA was prepared from E. coli cells by phenol-chloroform extraction, and ~10 µg of total RNA was used to synthesize cDNA. After purification with a Qiagen cDNA purification kit (Qiagen, Inc.), the cDNA was fragmented with DNase I. The fragmented cDNA was end biotinylated by terminal transferase with biotinylated ddATP.
|
| |
|
| Hybridization protocol |
Hybridization of the fragmented, biotinylated cDNA to a chip, washing, and staining of the chip was done on an Affymetrix workstation as described previously (Expression Analysis Technical Manual; Affymetrix, Inc., Santa Clara, Calif.). Detailed method is equivalent to that described by Soupene and colleagues: http://nature.berkeley.edu/%7eopaliy/papers/MG1655/MAprotocol.pdf
|
| Scan protocol |
Scanning of the chip were done on an Affymetrix workstation as described previously (Expression Analysis Technical Manual; Affymetrix, Inc., Santa Clara, Calif.).
|
| Description |
The sucrose and NaCl hyperosmotic treatment data were compared to the no treatment control data separately.
|
| Data processing |
Background correction, normalization and calculation of the expression measures were performed using RMA procedure with the WebArray platform (http://bioinformatics.skcc.org/webarray/). Subsequently the program EDGE (http://www.biostat.washington.edu/software/jstorey/edge/) was used to estimate the conditional false discovery rate (FDR), the expected proportion of false positives conditioned on having k 'significant' findings.
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| |
|
| Submission date |
Jul 21, 2008 |
| Last update date |
Jul 24, 2008 |
| Contact name |
John Phillip Bowman |
| E-mail(s) |
[email protected]
|
| Phone |
61 03 6226 6380
|
| Fax |
61 3 6226 2642
|
| Organization name |
University of Tasmania
|
| Department |
School of Agricultural Science
|
| Lab |
Australian Food Safety Centre of Excellence
|
| Street address |
College Road, Sandy Bay
|
| City |
Hobart |
| State/province |
Tasmania |
| ZIP/Postal code |
7005 |
| Country |
Australia |
| |
|
| Platform ID |
GPL3154 |
| Series (1) |
| GSE12190 |
Ion transport and osmotic adjustment in E. coli in response to ionic and non-ionic osmotica |
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