|
| Status |
Public on Aug 15, 2011 |
| Title |
Mtb_whiB5_15'_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Mtb TB84 15' induction
|
| Organism |
Mycobacterium tuberculosis H37Rv |
| Characteristics |
strain: TB84
|
| Treatment protocol |
MTb strains TB84 (containing the plasmid for whiB5 overexpression) and TB60 (containing the empty plasmid) were grown at 37°C in the presence of pristinamycin I 1 mg/ml for 15’ or 3 h and compared on whole genome DNA microarrays
|
| Growth protocol |
Bacteria were grown in Middlebrook 7H9 broth or 7H10 agar medium supplemented with 0.05% Tween 80, 0.2% glycerol and 10% ADN (Albumin, Dextrose, NaCl) at 37°C. When necessary streptomycin was added at 20 mg/ml. The inducer Pristinamycin I (Sanofi-Aventis, Paris, France) was dissolved in DMSO (50 mg/ml) and used at 1mg/ml. Liquid cultures were incubated in rolling bottles with gentle rotation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction from in vitro cultured M. tuberculosis was performed as previously described (Maciag et al., 2007) and analysed using the Agilent Bioanalyser 2100 system (Agilent Technologies) according to the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
Fluorescently labeled cDNA copies of total RNA were prepared by direct incorporation of fluorescent nucleotide analogues during a first-strand reverse transcription reaction as previously described (Provvedi et al., 2009).
|
| |
|
| Channel 2 |
| Source name |
Mtb TB60 15' induction
|
| Organism |
Mycobacterium tuberculosis H37Rv |
| Characteristics |
strain: TB60
|
| Treatment protocol |
MTb strains TB84 (containing the plasmid for whiB5 overexpression) and TB60 (containing the empty plasmid) were grown at 37°C in the presence of pristinamycin I 1 mg/ml for 15’ or 3 h and compared on whole genome DNA microarrays
|
| Growth protocol |
Bacteria were grown in Middlebrook 7H9 broth or 7H10 agar medium supplemented with 0.05% Tween 80, 0.2% glycerol and 10% ADN (Albumin, Dextrose, NaCl) at 37°C. When necessary streptomycin was added at 20 mg/ml. The inducer Pristinamycin I (Sanofi-Aventis, Paris, France) was dissolved in DMSO (50 mg/ml) and used at 1mg/ml. Liquid cultures were incubated in rolling bottles with gentle rotation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction from in vitro cultured M. tuberculosis was performed as previously described (Maciag et al., 2007) and analysed using the Agilent Bioanalyser 2100 system (Agilent Technologies) according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Fluorescently labeled cDNA copies of total RNA were prepared by direct incorporation of fluorescent nucleotide analogues during a first-strand reverse transcription reaction as previously described (Provvedi et al., 2009).
|
| |
|
| |
| Hybridization protocol |
Competitive hybridizations with equal amounts of purified Cy3/Cy5-labelled cDNA were performed in duplicate with both dye arrangements, as previously described (Provvedi et al., 2009).
|
| Scan protocol |
Microarrays were scanned using an Agilent G2565CA Microarray Scanner System (Scan Control Software 8.1) (Agilent technologies) and fluorescence intensities of the two channels at each spot were quantified using the Agilent Feature Extraction Software 10.1 (Agilent technologies).
|
| Description |
Biological replicate 1 of 4. 15' induction
|
| Data processing |
Data were normalized with a web-based tool for Diagnosis and Normalization of spotted cDNA MicroArrayData (DNMAD) (http://dnmad.bioinfo.cnio.es/) (Vaquerizas et al., 2005) using the print-tip lowess method after background subtraction. Genes significantly differentially expressed were identified using the Significance Analysis of Microarrays (SAM) tool (Tusher et al., 2001). Differentially expressed genes were defined by a q-value equal to 0% and a minimum fold difference of ±2 between the two samples. SAM is part of the TigerMultiExperiment Viewer package version 4.6 (TMeV) available at http://www.tm4.org/mev/
|
| |
|
| Submission date |
Jun 29, 2011 |
| Last update date |
Aug 15, 2011 |
| Contact name |
Stefano Casonato |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Padova
|
| Department |
Histology, microbiology and medical biotechnologies
|
| Lab |
Manganelli Riccardo
|
| Street address |
via A. Gabelli, 63
|
| City |
Padova |
| ZIP/Postal code |
35121 |
| Country |
Italy |
| |
|
| Platform ID |
GPL4057 |
| Series (1) |
| GSE30299 |
Identification of the WhiB5 regulon in Mycobacterium tuberculosis H37Rv |
|
