|
| Status |
Public on Mar 28, 2007 |
| Title |
Rv-SDS(5) vs Rv-D981[mpr]-SDS(5) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Rv treated with SDS
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
strain H37Rv
Rv treated with SDS for 90 min
|
| Treatment protocol |
Cultures were grown to early log phase (O.D.600 < 0.2), and divided into two 25 ml samples. SDS was added to one sample for each strain to a final concentration of 0.05%. Samples were incubated at 37oC, and then aliquots were removed at 2 hr or 5 hr, centrifuged to remove SDS, and then diluted in PBS prior to plating in duplicate. Coloines were counted after 2 weeks to determine percent survival in the stress-treated samples compared to untreated controls. No additional increase in colony number was observed after further incubation.
|
| Growth protocol |
M. tuberculosis mutants and parental strain H37Rv were grown in either Middlebrook 7H9 broth containing 0.05% Tween 80, or Middlebrook 7H10 agar (Difco), both enriched with 10% oleic acid-albumin-dextrose-catalase (OADC; Difco). Broth cultures were incubated at 37oC with gentle shaking, under normal atmospheric conditions (without additional CO2), except where indicated. Escherichia coli Novablue and Rosetta(DE3)pLysS (Novagen) were used as host strains for general cloning and gene expression, respectively. E. coli strains were grown on L agar or in L broth. Antibiotics were added to growth media as required.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mycobacterial strains were cultured in 7H9 medium to mid-log phase (OD600nm of 0.4-0.5) at 37oC with shaking. Cultures for the DNA microarray experiments were grown in the absence of CO2. For other experiments, cultures were grown with or without 5% CO2 as indicated. Total RNA was isolated using TRIzol LS Reagent (Invitrogen) according to the manufacturer's instruction, except that lysing matrix B and a FastPrep FP120 shaker (BIO 101) were used to disrupt the mycobacteria. With large culture volumes, bacteria were first pelleted by centrifugation and resuspended in a small volume of 0.2% Tween, prior to adding to the lysing tubes. Chromosomal DNA was removed with DNA-free reagents (Ambion), according to the manufacturer's instructions. For experiments with detergent stress, cultures were grown to log phase (O.D.600 of 0.3-0.4), SDS was added to a final concentration of 0.05%, and RNA was extracted from control and SDS-treated samples after 90 min incubation at 37?.
|
| Label |
Cy3
|
| Label protocol |
cDNA was synthesized using random primers and labeled with Cyanine-3 or Cyanine-5 dUTP (PerkinElmer, Wellesley, MA) by a modification of the procedure described by Voskuil et. al. {Voskuil, 2003 #137} and hybridized to the arrays overnight.
|
| |
|
| Channel 2 |
| Source name |
Mpr treated with SDS
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
mpr mutant (Rv-D981)
Time: Mpr treated with SDS for 90 min
|
| Treatment protocol |
Cultures were grown to early log phase (O.D.600 < 0.2), and divided into two 25 ml samples. SDS was added to one sample for each strain to a final concentration of 0.05%. Samples were incubated at 37oC, and then aliquots were removed at 2 hr or 5 hr, centrifuged to remove SDS, and then diluted in PBS prior to plating in duplicate. Coloines were counted after 2 weeks to determine percent survival in the stress-treated samples compared to untreated controls. No additional increase in colony number was observed after further incubation.
|
| Growth protocol |
M. tuberculosis mutants and parental strain H37Rv were grown in either Middlebrook 7H9 broth containing 0.05% Tween 80, or Middlebrook 7H10 agar (Difco), both enriched with 10% oleic acid-albumin-dextrose-catalase (OADC; Difco). Broth cultures were incubated at 37oC with gentle shaking, under normal atmospheric conditions (without additional CO2), except where indicated. Escherichia coli Novablue and Rosetta(DE3)pLysS (Novagen) were used as host strains for general cloning and gene expression, respectively. E. coli strains were grown on L agar or in L broth. Antibiotics were added to growth media as required.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mycobacterial strains were cultured in 7H9 medium to mid-log phase (OD600nm of 0.4-0.5) at 37oC with shaking. Cultures for the DNA microarray experiments were grown in the absence of CO2. For other experiments, cultures were grown with or without 5% CO2 as indicated. Total RNA was isolated using TRIzol LS Reagent (Invitrogen) according to the manufacturer's instruction, except that lysing matrix B and a FastPrep FP120 shaker (BIO 101) were used to disrupt the mycobacteria. With large culture volumes, bacteria were first pelleted by centrifugation and resuspended in a small volume of 0.2% Tween, prior to adding to the lysing tubes. Chromosomal DNA was removed with DNA-free reagents (Ambion), according to the manufacturer's instructions. For experiments with detergent stress, cultures were grown to log phase (O.D.600 of 0.3-0.4), SDS was added to a final concentration of 0.05%, and RNA was extracted from control and SDS-treated samples after 90 min incubation at 37?.
|
| Label |
Cy5
|
| Label protocol |
cDNA was synthesized using random primers and labeled with Cyanine-3 or Cyanine-5 dUTP (PerkinElmer, Wellesley, MA) by a modification of the procedure described by Voskuil et. al. {Voskuil, 2003 #137} and hybridized to the arrays overnight.
|
| |
|
| |
| Hybridization protocol |
Slides were blocked prior to hybridization using BSA The purified cDNA probes were added to the arrays together with a hybridization solution mix resulting in a final concentration of 0.5ug/ul tRNA (Invitrogen), 2.0X SSC (Ambion), 0.25% Formamide (Sigma) and 0.1% SDS (Ambion). The probes were covered by a flat 22x22mm coverslip (Corning) and hybridized in sealed hybridization chambers (Genemachines) for sixteen hours in a 50oC water bath.
|
| Scan protocol |
The slides were scanned using an Axon 4000B scanner
|
| Description |
Rv-SDS(5) vs Rv-D981[mpr]-SDS(5)
|
| Data processing |
The images were processed using GenePix 5.1 and resulting text files were exported to Microsoft Excel
|
| |
|
| Submission date |
Jan 16, 2007 |
| Last update date |
Mar 28, 2007 |
| Contact name |
Saleena Ghanny |
| E-mail(s) |
[email protected]
|
| Organization name |
Rutgers University
|
| Street address |
185 South Orange Ave
|
| City |
Newark |
| ZIP/Postal code |
07103 |
| Country |
USA |
| |
|
| Platform ID |
GPL4057 |
| Series (1) |
| GSE6750 |
Complex interactions of stress-associated regulons in an mprAB deletion mutant of Mycobacterium tuberculosis |
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