|
| Status |
Public on May 24, 2010 |
| Title |
Aerobic peroxynitrite chemostat culture 3. Slide 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Steady-state continuous cultured control aerobic samples in presence of 300uM peroxynitrite for 5 minutes
|
| Organism |
Escherichia coli |
| Characteristics |
stress: 300uM peroxynitrite
strain: Wild type strain MG1655
|
| Treatment protocol |
At steady-state, peroxynitrite was added to the chemostat culture and to the nutrient feed at a final concentration of 300 uM. Samples were removed immediately prior to and 5 minutes after injection of peroxynitrite
|
| Growth protocol |
Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow III Biofermentor under continuous culture (chemostat) conditions. Cells were grown in defined media containing 2 mM glycerol as the sole and limiting source of energy and carbon. The working volume was 1 litre, and the dilution rate 0.2 h-1. For aerobic growth, the air-flow rate was 1 l/min, and the dissolved oxygen tension was maintained at 40% air saturation
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples (10 ml) were harvested directly into RNA Protect (Qiagen) to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers. RNA was quantified using a BioPhotometer (Eppendorf).
|
| Label |
Cy3
|
| Label protocol |
The labelling procedure uses equal quantities of RNA from adequate hydrogen peroxide and control samples
Approximately 9 µg RNA was annealed to 4.5 µg pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC and cooling on ice for 2 min. This was supplemented with 6 ml of 5× First-strand buffer (Invitrogen), 2.5 ml dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 3 ml 0.1 M DTT, 2 ml 1 mM Cy5 (Perkin Elmer) and 1.5 ml Superscript III reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 3 hours. The reaction was terminated by the addition of NaOH (7.5 ml of 1M) and HCl (7.5 ml of 1M) before the addition of TE buffer (300 ml). Labelled cDNA was purified using a Qiaquick purification kit (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
Steady-state continuous cultured control aerobic samples in absence of peroxynitrite
|
| Organism |
Escherichia coli |
| Characteristics |
stress: control
strain: Wild type strain MG1655
|
| Treatment protocol |
At steady-state, peroxynitrite was added to the chemostat culture and to the nutrient feed at a final concentration of 300 uM. Samples were removed immediately prior to and 5 minutes after injection of peroxynitrite
|
| Growth protocol |
Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow III Biofermentor under continuous culture (chemostat) conditions. Cells were grown in defined media containing 2 mM glycerol as the sole and limiting source of energy and carbon. The working volume was 1 litre, and the dilution rate 0.2 h-1. For aerobic growth, the air-flow rate was 1 l/min, and the dissolved oxygen tension was maintained at 40% air saturation
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples (10 ml) were harvested directly into RNA Protect (Qiagen) to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers. RNA was quantified using a BioPhotometer (Eppendorf).
|
| Label |
Cy5
|
| Label protocol |
The labelling procedure uses equal quantities of RNA from adequate hydrogen peroxide and control samples
Approximately 9 µg RNA was annealed to 4.5 µg pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC and cooling on ice for 2 min. This was supplemented with 6 ml of 5× First-strand buffer (Invitrogen), 2.5 ml dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 3 ml 0.1 M DTT, 2 ml 1 mM Cy5 (Perkin Elmer) and 1.5 ml Superscript III reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 3 hours. The reaction was terminated by the addition of NaOH (7.5 ml of 1M) and HCl (7.5 ml of 1M) before the addition of TE buffer (300 ml). Labelled cDNA was purified using a Qiaquick purification kit (Qiagen).
|
| |
|
| |
| Hybridization protocol |
The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample and re-suspended in 120 ml salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95 oC for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 °C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 °C) SSC buffers for 5 min each at 37oC: 1× SSC/0.1 % SDS, 1× SSC, 0.2× SSC and 0.01× SSC. Microarray slides were dried by centrifugation at 500 × g for 2 min before scanning.
|
| Scan protocol |
Slides were scanned on an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5).
|
| Description |
no additional information
|
| Data processing |
Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values.
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| |
|
| Submission date |
Dec 08, 2009 |
| Last update date |
May 24, 2010 |
| Contact name |
Robert Poole |
| E-mail(s) |
[email protected]
|
| Phone |
01142224447
|
| Organization name |
University of Sheffield
|
| Department |
Molecular Biology & Biotechnology
|
| Lab |
F13
|
| Street address |
Firth Court, Western Bank
|
| City |
Sheffield |
| State/province |
South Yorkshire |
| ZIP/Postal code |
S10 2TN |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL534 |
| Series (1) |
| GSE19370 |
Transcriptional profiling of E. coli after addition of peroxynitrite or hydrogen peroxide to aerobically growing cells |
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