|
| Status |
Public on Aug 01, 2007 |
| Title |
Shifted_from_20%_oxygen_to_0.2%_and_grew_by_2_hours |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Cell culture grew in 20% oxygen
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
H37Rv (ATCC 27294)
|
| Treatment protocol |
Frozen cell pellets were suspended in 1 ml of Trizol reagent (GIBCOy BRL) and transferred to 2-ml screw-cap tubes containing 0.4-ml of 0.1-mm diameter zirconiaysilica beads (BioSpec Products, Bartlesville, OK).
|
| Growth protocol |
H37Rv (ATCC 27294) and Mycobacterium bovis bacillus Calmette–Gue´rin (BCG) Montreal (ATCC 35735) were maintained on Middlebrook 7H9 medium (Difco) with 0.05% Tween 80 and ADC supplement or on Middlebrook 7H10 plates at 37 C. Bacilli were grown in roller bottles unless otherwise noted. When needed, kanamycin was used at 30 ug/ml and hygromycin at 50 ug/ml. Defined hypoxic culture was as previously described. Gas flow was maintained at aproximatly at 0.15 standard cubic feet (4.2 liters) per hour within each flask.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were disrupted by three 30-sec pulses in a BioSpec Products bead beater. After 5 min at room temperature, samples were centrifuged at 13,000 x g for 45 sec, and the supernatants were extracted with 300 ul of chloroformyisoamyl alcohol (24:1) and precipitated with isopropyl alcohol. Samples were incubated 10 min to overnight at 4 C and centrifuged for 10 min at 4 C. The RNA pellets were washed with 1 ml of 75% ethanol, centrifuged at 13,000 x g for 5 min, and air-dried. After suspension of the RNA pellets in 90 ul of water, 10 ul of DNase I 10x buffer and 4 units of DNase I (Ambion, Austin, TX) were added and the samples were incubated for 30 min. Final purification of RNA was by RNeasy column (Qiagen, Chatsworth, CA).
|
| Label |
Cy3
|
| Label protocol |
All steps in the MTB DNA microarray gene expression analysis were performed as described (PMID: 10536008 and 11398407). Controls for array quality were printed onto each slide and included a dilution series of MTB genomic DNA (positive control) and salmon sperm DNA (negative control). Additionally, DNA prelabeled with the cyanine dyes Cy3 or Cy5 were printed to ascertain whether spot-to-spot carryover contamination had occurred during the printing step. The quality of cDNA labeling was monitored by quantitatively measuring the intensities of Cy3- and Cy5-labeled samples hybridized to the MTB genomic positive control spots.
|
| |
|
| Channel 2 |
| Source name |
Cell culture shifted from 20% oxygen to 0.2% and grew by 2 hours
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
H37Rv (ATCC 27294)
|
| Treatment protocol |
Frozen cell pellets were suspended in 1 ml of Trizol reagent (GIBCOy BRL) and transferred to 2-ml screw-cap tubes containing 0.4-ml of 0.1-mm diameter zirconiaysilica beads (BioSpec Products, Bartlesville, OK).
|
| Growth protocol |
H37Rv (ATCC 27294) and Mycobacterium bovis bacillus Calmette–Gue´rin (BCG) Montreal (ATCC 35735) were maintained on Middlebrook 7H9 medium (Difco) with 0.05% Tween 80 and ADC supplement or on Middlebrook 7H10 plates at 37 C. Bacilli were grown in roller bottles unless otherwise noted. When needed, kanamycin was used at 30 ug/ml and hygromycin at 50 ug/ml. Defined hypoxic culture was as previously described. Gas flow was maintained at aproximatly at 0.15 standard cubic feet (4.2 liters) per hour within each flask.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were disrupted by three 30-sec pulses in a BioSpec Products bead beater. After 5 min at room temperature, samples were centrifuged at 13,000 x g for 45 sec, and the supernatants were extracted with 300 ul of chloroformyisoamyl alcohol (24:1) and precipitated with isopropyl alcohol. Samples were incubated 10 min to overnight at 4 C and centrifuged for 10 min at 4 C. The RNA pellets were washed with 1 ml of 75% ethanol, centrifuged at 13,000 x g for 5 min, and air-dried. After suspension of the RNA pellets in 90 ul of water, 10 ul of DNase I 10x buffer and 4 units of DNase I (Ambion, Austin, TX) were added and the samples were incubated for 30 min. Final purification of RNA was by RNeasy column (Qiagen, Chatsworth, CA).
|
| Label |
Cy5
|
| Label protocol |
All steps in the MTB DNA microarray gene expression analysis were performed as described (PMID: 10536008 and 11398407). Controls for array quality were printed onto each slide and included a dilution series of MTB genomic DNA (positive control) and salmon sperm DNA (negative control). Additionally, DNA prelabeled with the cyanine dyes Cy3 or Cy5 were printed to ascertain whether spot-to-spot carryover contamination had occurred during the printing step. The quality of cDNA labeling was monitored by quantitatively measuring the intensities of Cy3- and Cy5-labeled samples hybridized to the MTB genomic positive control spots.
|
| |
|
| |
| Hybridization protocol |
All steps in the MTB DNA microarray gene expression analysis were performed as described (PMID: 10536008 and 11398407).
|
| Scan protocol |
Microarrays were scanned using a GenePix 4000 A (Axon Instruments). The intensities of the two dyes at each spot were quantified using SCANALYZE (M. Eisen; http://rana.lbl.gov/EisenSoftware.htm).
|
| Description |
Biological replicate 1 of 6. Control, strain H37Rv. Cell culture grew in 20% oxygen.
|
| Data processing |
The overall reproducibility of the microarray experiments was evaluated using the Signifi-cance Analysis of Microarrays (SAM) program. The SAM algorithm was set for two-class unpaired analysis with 500 permutations and K-nearest imputer for missing data. Significantly regulated genes were selected by adjusting the delta value to give a false discovery rate below 1%. In each data set, all genes regulated 1.5-fold and greater were determined to be significant with a false discovery rate of less than 1%, indicating a high degree of reproducibility.
|
| |
|
| Submission date |
Jul 31, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
SMD Staff |
| E-mail(s) |
array@genome.stanford.edu
|
| Phone |
650-498-6012
|
| URL |
http://genome-www5.stanford.edu/
|
| Organization name |
Stanford Microarray Database (SMD)
|
| Department |
Stanford University, School of Medicine
|
| Street address |
300 Pasteur Drive
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL2787 |
| Series (1) |
| GSE8639 |
Regulation of the Mycobacterium tuberculosishypoxic response gene encoding alpha-crystallin |
|
