|
| Status |
Public on Jun 26, 2009 |
| Title |
dosSTKO_24_hour_Gaspak_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
ΔdosS/T, 24 hours gaspak treatment
|
| Organism |
Mycobacterium tuberculosis H37Rv |
| Characteristics |
strain: H37Rv
mutation: ΔdosS/T
condition: 24 hours in anaerobic chamber with anaerobic envelope
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets were flash frozen on dry ice, resuspended in Trizol, and bead-beaten 3 x 40 seconds. Resulting cell lysate was added to chloroform and nucleic acids were then precipitated in isopropanol, rehydrated in 75% ethanol, then DNAse treated. Fur further purification, Qiagen RNeasy kit was used according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
1.5 ug of RNA was primed with N6 primers, heated for two minutes at 98 C, then incubated 10 minutes at 25 C followed by 90 minutes at 42 C. 5mM A,G,C and 0.2mM T dNTP mix was used.
|
| |
|
| Channel 2 |
| Source name |
wild type, 24 hours gaspak treatment
|
| Organism |
Mycobacterium tuberculosis H37Rv |
| Characteristics |
strain: H37Rv
mutation: wild type
condition: 24 hours in anaerobic chamber with anaerobic envelope
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets were flash frozen on dry ice, resuspended in Trizol, and bead-beaten 3 x 40 seconds. Resulting cell lysate was added to chloroform and nucleic acids were then precipitated in isopropanol, rehydrated in 75% ethanol, then DNAse treated. Fur further purification, Qiagen RNeasy kit was used according to the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
1.5 ug of RNA was primed with N6 primers, heated for two minutes at 98 C, then incubated 10 minutes at 25 C followed by 90 minutes at 42 C. 5mM A,G,C and 0.2mM T dNTP mix was used.
|
| |
|
| |
| Hybridization protocol |
Arrays were prehybridized in water, SSC, BSA, and SDS. Labeling reaction was concentrated to 5ul in a microcon 10 filter tube, added to tRNA, SSC, formamide, and SDS, and heated to 98 C for two minutes. Samples were hybridzed overnight at 50 C.
|
| Scan protocol |
Scanned on Molecular Device's Genepix 4000B.
|
| Description |
TVB211
|
| Data processing |
Spot intensities were obtained using GenePix Pro 6.0. Data analysis was performed as previously described in PMID: 12953092 (Voskuil M, et al., J Exp Med. 2003 Sep 1;198(5):705-13).
|
| |
|
| Submission date |
Jun 25, 2009 |
| Last update date |
Jun 25, 2009 |
| Contact name |
Ryan William Honaker |
| Organization name |
University of Colorado Denver
|
| Department |
Microbiology
|
| Lab |
Marty Voskuil
|
| Street address |
12800 E. 19th Ave.
|
| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
| |
|
| Platform ID |
GPL8768 |
| Series (1) |
| GSE16811 |
The unique roles of DosT and DosS in DosR regulon induction and Mycobacterium tuberculosis dormancy |
|
