|
| Status |
Public on Dec 17, 2009 |
| Title |
M.bovis-NO vs M.bovis_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
M. bovis-NO culture
|
| Organism |
Mycobacterium tuberculosis variant bovis |
| Characteristics |
strain: Ravenel (TMC 401)
treatment: nitric oxide
|
| Biomaterial provider |
Public Health Research Institute, Newark,NJ.
|
| Treatment protocol |
Mid-log phase cultures were exposed to 50 uM diethylenetriamine/nitric oxide adduct (DETA/NO) for 40 minutes.
|
| Growth protocol |
Culture grew in Middlebrook 7H9 media supplemented with ADS, pyruvate and Tween 80.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After NO exposure, culture was shortly centrifuged and the pellet quickly frozen with dry ice. RNA was extracted according to published protocols.
Manganelli et al. Mol Microbiol (2001) 41(2):423-437
|
| Label |
Cy3
|
| Label protocol |
Walters et al. Mol Microbiol (2006) 60(2):312-330
|
| |
|
| Channel 2 |
| Source name |
M. bovis culture
|
| Organism |
Mycobacterium tuberculosis variant bovis |
| Characteristics |
strain: Ravenel (TMC 401)
treatment: none
|
| Biomaterial provider |
Public Health Research Institute, Newark,NJ.
|
| Treatment protocol |
No treatment
|
| Growth protocol |
Culture grew in Middlebrook 7H9 media supplemented with ADS, pyruvate and Tween 80.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mid-log phase culture was shortly centrifuged and the pellet quickly frozen with dry ice. RNA was extracted according to published protocols.
Manganelli et al. Mol Microbiol (2001) 41(2):423-437
|
| Label |
Cy5
|
| Label protocol |
Walters et al. Mol Microbiol (2006) 60(2):312-330
|
| |
|
| |
| Hybridization protocol |
Total purified cDNA was added to the arrays in a hybridization solution containing a final concentration of 0.5 ug/ul tRNA, 2.0x SSC, 0.25% formamide and 0.1% SDS. For each array, cDNA prepared from the M. tuberculosis RNA was mixed with cDNA from M. bovis. The slides were covered by a flat 22 x 22 mm coverslip and hybridized in sealed hybridization chambers for sixteen hours at 50°C in a water bath.
|
| Scan protocol |
Prepared slides were immediately scanned using an Axon 4000A Scanner (Axon Instruments). Fluorescence intensities of Cy3 and Cy5 dies at each spot were quantified using the GenePix Pro 4.0 software.
|
| Description |
Synthesis and hybridization of labelled samples was performed as described in previously published protocols (Walters et al. Mol Microbiol (2006) 60(2):312-330).
|
| Data processing |
Intensity values obtained from spots on the chip were corrected by background subtraction. Corrected data were then normalized using the Lowess implementation included in GeneSpring 7.2 software (Silicon Genetics). The values are expressed as log2 (NO/untreated) ratios determined from the normalized fluorescence intensity.
|
| |
|
| Submission date |
Mar 09, 2009 |
| Last update date |
Dec 16, 2009 |
| Contact name |
Germán Rehren |
| E-mail(s) |
grehren@uach.cl
|
| Organization name |
Universidad Austral de Chile
|
| Department |
Biochemistry
|
| Lab |
Prof. Zárraga
|
| Street address |
Campus Isla Teja
|
| City |
Valdivia |
| State/province |
Valdivia |
| ZIP/Postal code |
5090000 |
| Country |
Chile |
| |
|
| Platform ID |
GPL4057 |
| Series (1) |
| GSE15164 |
Mycobacterium bovis transcriptional response to nitric oxide |
|
