|
| Status |
Public on Jul 31, 2013 |
| Title |
M982THB_r2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
M982 bacteria cultured in THB medium during 1h
|
| Organism |
Neisseria meningitidis |
| Characteristics |
strain: M982
serotype: B
medium: THB
time: 1 hour
|
| Treatment protocol |
Cultures were harvested by centrifugation for 20 min (3000 g, 4°C), and pellets were stored at -80°C.
|
| Growth protocol |
Strains MC58 and M982 were grown on BHI (brain-heart infusion [Difco]) agar or GC (gonococcal culture [Difco]) agar supplemented with 1% Vitox (Oxoid), at 37°C with 10% CO2 overnight. Bacteria were harvested, resuspended in D-PBS (Dulbecco’s phosphate-buffered saline [Gibco]) and then used to inoculate 50 ml of liquid growth medium in baffled shake flasks at initial OD600 of 0.25. Bacteria previously grown on plates of BHI agar or GC agar were inoculated, respectively, in THB (Todd Hewitt Broth [Difco]) or in the modified Catlin medium MC.2. Cultures were incubated at 37°C in a shaking incubator for 1h or 7h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using RNAwiz (Ambion) according to the manufacturer specifications with slight modifications.
|
| Label |
Cy3,Cy5
|
| Label protocol |
For each set of conditions, 20 µg of RNA were labeled differentially with Cy3-dCTP and Cy5-dCTP (Amersham Biosciences) in a first-strand Reverse Transcription reaction using Superscript II RNase H RT (Invitrogen).
|
| |
|
| Channel 2 |
| Source name |
M982 bacteria cultured in THB medium during 7h
|
| Organism |
Neisseria meningitidis |
| Characteristics |
strain: M982
serotype: B
medium: THB
time: 7 hours
|
| Treatment protocol |
Cultures were harvested by centrifugation for 20 min (3000 g, 4°C), and pellets were stored at -80°C.
|
| Growth protocol |
Strains MC58 and M982 were grown on BHI (brain-heart infusion [Difco]) agar or GC (gonococcal culture [Difco]) agar supplemented with 1% Vitox (Oxoid), at 37°C with 10% CO2 overnight. Bacteria were harvested, resuspended in D-PBS (Dulbecco’s phosphate-buffered saline [Gibco]) and then used to inoculate 50 ml of liquid growth medium in baffled shake flasks at initial OD600 of 0.25. Bacteria previously grown on plates of BHI agar or GC agar were inoculated, respectively, in THB (Todd Hewitt Broth [Difco]) or in the modified Catlin medium MC.2. Cultures were incubated at 37°C in a shaking incubator for 1h or 7h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using RNAwiz (Ambion) according to the manufacturer specifications with slight modifications.
|
| Label |
Cy5,Cy3
|
| Label protocol |
For each set of conditions, 20 µg of RNA were labeled differentially with Cy3-dCTP and Cy5-dCTP (Amersham Biosciences) in a first-strand Reverse Transcription reaction using Superscript II RNase H RT (Invitrogen).
|
| |
|
| |
| Hybridization protocol |
The Cy3- and Cy5-specific cDNAs were combined and purified using the QIAquick PCR purification kit (QIAGEN) according to the manufacturer specifications. The labeled cDNAs were concentrated using Microcon YM-30 filter units (Millipore) and then vacuum concentrated. The samples were heated to 95°C for 2 min and briefly cooled before addition of 1 volume of Microarray hybridization buffer (Amersham) and 2 volumes of deionized formamide (Sigma Aldrich). The labeled cDNA samples were hybridized to the array at 42°C for 16h.
|
| Scan protocol |
Slides were scanned with a GenePix Personal 4100A scanner (Axon Instruments). The signal intensities were measured with the GenePix PRO v6.1 software.
|
| Description |
Analysis used bacteria cultured in different media at different times.
M982 bacteria cultured in THB medium during 1h versus 7h.
|
| Data processing |
Data analysis were carried out with Rosetta Resolver software v4 (Rosetta biosoftware). Data normalization and transformation was performed with the Axon/GenePix error model when loading data into Rosetta Resolver. All experiments were performed in dye swap.
|
| |
|
| Submission date |
Dec 15, 2010 |
| Last update date |
Jul 31, 2013 |
| Contact name |
Bachra Rokbi |
| E-mail(s) |
[email protected]
|
| Organization name |
sanofi pasteur
|
| Street address |
1541 Av M Mérieux
|
| City |
Marcy l'Etoile |
| ZIP/Postal code |
69280 |
| Country |
France |
| |
|
| Platform ID |
GPL11321 |
| Series (1) |
| GSE26078 |
Global transcriptomic response of serum-resistant Neisseria meningitidis |
|
