|
| Status |
Public on Apr 27, 2013 |
| Title |
16MΔhfq(CY5)-16M(CY3), rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Brucella-16MΔhfq-GEM 4.0
|
| Organism |
Brucella melitensis |
| Characteristics |
strain/background: 16M
genotype: Δhfq
|
| Treatment protocol |
Bacteria was transferred from TSB to GEM 4.0 (limited nutrition and acid) for 30min.
|
| Growth protocol |
16M and 16MΔhfq strains were grown in TSB at 37℃ to the stationary phase, and then transferred to the stress condition where hfq was greatly activated.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from liquid cultures of B. melitensis using Trizol reagent (Invitrogen) as recommended by the manufacturer. RNA samples were then treated with DNase I (Promega) to remove any contaminating genomic DNA.
|
| Label |
Cy5
|
| Label protocol |
RNA was reverse transcribed into cDNA in the present of aminoallyl-dUTP, and then labeled with Cy3 or Cy5 monofunctional dye (Amersham).
|
| |
|
| Channel 2 |
| Source name |
16M cultured in GEM 4.0 for 30min
|
| Organism |
Brucella melitensis |
| Characteristics |
strain/background: 16M
genotype: wild type
|
| Treatment protocol |
Bacteria was transferred from TSB to GEM 4.0 (limited nutrition and acid) for 30min.
|
| Growth protocol |
16M and 16MΔhfq strains were grown in TSB at 37℃ to the stationary phase, and then transferred to the stress condition where hfq was greatly activated.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from liquid cultures of B. melitensis using Trizol reagent (Invitrogen) as recommended by the manufacturer. RNA samples were then treated with DNase I (Promega) to remove any contaminating genomic DNA.
|
| Label |
Cy3
|
| Label protocol |
RNA was reverse transcribed into cDNA in the present of aminoallyl-dUTP, and then labeled with Cy3 or Cy5 monofunctional dye (Amersham).
|
| |
|
| |
| Hybridization protocol |
The slides were pre-hybridized in a buffer containing 5×SSC, 0.1% SDS and 0.1% BSA, and then washed and blown to dry by hot air. The Cy3/Cy5 differentially labeled cDNA samples were resuspended in hybridization solution (50% deionized formamide, 5×SSC, 0.1% SDS, 5×Denhardt's solution, and 0.5μg/μl of sheared salmon sperm DNA), and then hybridized with the slides at 42℃ for 18-20 h. After hybridization, the slides were washed in 1×SSC with 0.06% SDS for 2 min, then in 0.06×SSC for 2 min and finally in ethanol for 2 min.
|
| Scan protocol |
The slides were blown to dry by hot air and were scanned by using a GenePix Personal 4100A Microarray Scanner.
|
| Description |
Biological replicate 3 of 4
|
| Data processing |
The scanning images were processed and the data were further analyzed by using GenePix Pro 4.1 software (Axon Instruments) combined with Microsoft Excel software. Spots were analyzed by adaptive quantitation, and the local background was subsequently subtracted. Spots with background-corrected signal intensity (median) in both channels lower than 2-fold of background intensity (median) were rejected from further analysis, and then the remaining data points were normalized by total intensity normalization methods. The normalized log2 ratio of test/reference signal for each spot was recorded. The averaged log2 ratio for each gene with at least four data points was finally calculated.
|
| |
|
| Submission date |
Apr 26, 2013 |
| Last update date |
Apr 27, 2013 |
| Contact name |
tai 2006 |
| E-mail(s) |
[email protected]
|
| Organization name |
Department of Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences
|
| Street address |
Dongdajie
|
| City |
Beijing |
| ZIP/Postal code |
100071 |
| Country |
China |
| |
|
| Platform ID |
GPL11117 |
| Series (1) |
| GSE46418 |
Comparative transcriptome analysis of Brucella melitensis 16M and its hfq mutant under acidified minimum medium (GEM4.0) conditions |
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