|
| Status |
Public on Aug 04, 2007 |
| Title |
Exposed to Diamide 5 mM sigH mutant rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Cell culture untreated
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
sigH mutant (H37Rv sigH::hyg)
|
| 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 |
All the experiments were performed with M. tuberculosis H37Rv and its derivatives obtained during this study. Bacteria were grown in either Middlebrook 7H9 (liquid medium) or 7H10 (solid medium; Difco) both supplemented with ADN, 0.2% glycerol and 0.05% Tween 80. Liquid cultures were grown in roller bottles at 37 C. Plates were incubated at 37 C in sealed plastic bags. Escherichia coli strain JM109 was grown in Luria broth (LB; Difco) at 37 C in shaking cultures. When required, antibiotics were added at the following concentrations: kanamycin, 20 ug/ml for M. tuberculosis or 50 ug/ml for E. coli; hygromycin, 150 ug/ml for M. tuberculosis or 50 ug/ml for E. coli; ampicillin, 100 ug/ml; streptomycin, 20 ug/ml. Sucrose selection was performed on 7H10 plates with 8% sucrose.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction for RTÐPCR experiments was performed as described previously. Briefly, bacteria were thawed on ice, broken with zirconia beads
(Biospec Products), extracted twice with phenolÐchloroformÐ isoamyl alcohol (25:24:1) and then precipitated in ethanol. To remove the chromosomal DNA, samples were treated twice with TRI reagent (Molecular Research Center) according to the manufacturerÕs instructions. RNA extraction for microarray experiments was performed using the following protocol: the frozen cell pellets were suspended in 1ml of Trizol reagent (Gibco BRL) and transferred to 2ml screw cap tubes containing 0.5 ml of 0.1-mm-diameter zirconia/silica beads (BioSpec Products). Cells were disrupted by two 30 s pulses in a BioSpec Products bead beater. After a 5 min incubation at room temperature, samples were centrifuged at maximum speed for 45 s, and the supernatants were transferred to 2ml Heavy Phase Lock Gel I tubes (Eppendorf) containing 300 ul of chloroformÐisoamyl alcohol (24:1), inverted rapidly for 15 s and incubated 2 min. The samples were centrifuged for 5 min, and the aqueous phase was added to 270 ul of isopropanol. After the addition of 270 ul of 0.8M Na citrate-1.2M NaCl, samples were incubated overnight at 4 C and centrifuged for 10 min at 4 C. The RNA pellets were washed with 1ml of 75% ethanol, centrifuged 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) were added, and the samples were incubated for 30 min. Final purification of RNA used an RNeasy column (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
All steps in the MTB DNA microarray gene expression analysis were performed as described. 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.
|
| |
|
| Channel 2 |
| Source name |
Cell culture exposed to Diamide 5 mM for 60 minutes
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
sigH mutant (H37Rv sigH::hyg)
|
| 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 |
All the experiments were performed with M. tuberculosis H37Rv and its derivatives obtained during this study. Bacteria were grown in either Middlebrook 7H9 (liquid medium) or 7H10 (solid medium; Difco) both supplemented with ADN, 0.2% glycerol and 0.05% Tween 80. Liquid cultures were grown in roller bottles at 37 C. Plates were incubated at 37 C in sealed plastic bags. Escherichia coli strain JM109 was grown in Luria broth (LB; Difco) at 37 C in shaking cultures. When required, antibiotics were added at the following concentrations: kanamycin, 20 ug/ml for M. tuberculosis or 50 ug/ml for E. coli; hygromycin, 150 ug/ml for M. tuberculosis or 50 ug/ml for E. coli; ampicillin, 100 ug/ml; streptomycin, 20 ug/ml. Sucrose selection was performed on 7H10 plates with 8% sucrose.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction for RTÐPCR experiments was performed as described previously. Briefly, bacteria were thawed on ice, broken with zirconia beads
(Biospec Products), extracted twice with phenolÐchloroformÐ isoamyl alcohol (25:24:1) and then precipitated in ethanol. To remove the chromosomal DNA, samples were treated twice with TRI reagent (Molecular Research Center) according to the manufacturerÕs instructions. RNA extraction for microarray experiments was performed using the following protocol: the frozen cell pellets were suspended in 1ml of Trizol reagent (Gibco BRL) and transferred to 2ml screw cap tubes containing 0.5 ml of 0.1-mm-diameter zirconia/silica beads (BioSpec Products). Cells were disrupted by two 30 s pulses in a BioSpec Products bead beater. After a 5 min incubation at room temperature, samples were centrifuged at maximum speed for 45 s, and the supernatants were transferred to 2ml Heavy Phase Lock Gel I tubes (Eppendorf) containing 300 ul of chloroformÐisoamyl alcohol (24:1), inverted rapidly for 15 s and incubated 2 min. The samples were centrifuged for 5 min, and the aqueous phase was added to 270 ul of isopropanol. After the addition of 270 ul of 0.8M Na citrate-1.2M NaCl, samples were incubated overnight at 4 C and centrifuged for 10 min at 4 C. The RNA pellets were washed with 1ml of 75% ethanol, centrifuged 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) were added, and the samples were incubated for 30 min. Final purification of RNA used an RNeasy column (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
All steps in the MTB DNA microarray gene expression analysis were performed as described. 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.
|
| |
|
| |
| Hybridization protocol |
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.
|
| 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 4 of 6. Cell culture exposed to Diamide 5 mM for 60 minutes. sigH mutant
|
| 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 |
Aug 03, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
SMD Staff |
| E-mail(s) |
[email protected]
|
| 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) |
| GSE8689 |
Role of the extracytoplasmic-function sigma Factor sigmaH in Mycobacterium tuberculosis global gene expression |
|
