|
| Status |
Public on Sep 22, 2009 |
| Title |
72 hour phosphate starvation replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Log phase culture
|
| Organism |
Mycobacterium tuberculosis CDC1551 |
| Characteristics |
Mycobacterium tuberculosis CDC1551 was grown in modified Middlebrook broth containing 25mM phosphate until log phase (OD600 ~0.4).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Log phase culture was centrifuged at 3500 rpm for 10 minutes at 4˚C. The supernatants were removed and the pellets resuspended in 1 ml TriZOL reagent (GIBCO/BRL). Mycobacterial membranes were disrupted using 0.1 mm zirconia/silica beads in a Bio-Spec bead beater (8 cycles x 30 sec/cycle). Mycobacterial RNA was recovered by centrifugation, chloroform extraction, and isopropyl alcohol precipitation. Prior to reverse transcription, RNA was treated with RNase-free DNase (Ambion) and subjected to 40 cycles of PCR to assure that all DNA had been removed, as assessed by ethidium bromide-stained agarose gel analysis.
|
| Label |
Cy5
|
| Label protocol |
Fluorescently-labeled cDNA was generated using Superscript III (Biorad) and fluorescent dyes Cy5 (Amersham).
|
| |
|
| Channel 2 |
| Source name |
72 hour phosphate-starved culture
|
| Organism |
Mycobacterium tuberculosis CDC1551 |
| Characteristics |
Mycobacterium tuberculosis CDC1551 was grown in modified Middlebrook broth containing 25 mM phosphate until log phase (OD600 ~0.4). Then culture was pelleted, washed twice in modified Middlebrook phosphate-free broth by centrifugation at 3000 rpm for 5 min and resuspended in an equal volume of phosphate-free broth. Phosphate-starved cultures were incubated for 72 hours in a shaker at 37 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
72 hour phosphate-starved culture was centrifuged at 3500 rpm for 10 minutes at 4˚C. The supernatants were removed and the pellets resuspended in 1 ml TriZOL reagent (GIBCO/BRL). Mycobacterial membranes were disrupted using 0.1 mm zirconia/silica beads in a Bio-Spec bead beater (8 cycles x 30 sec/cycle). Mycobacterial RNA was recovered by centrifugation, chloroform extraction, and isopropyl alcohol precipitation. Prior to reverse transcription, RNA was treated with RNase-free DNase (Ambion) and subjected to 40 cycles of PCR to assure that all DNA had been removed, as assessed by ethidium bromide-stained agarose gel analysis.
|
| Label |
Cy3
|
| Label protocol |
Fluorescently-labeled cDNA was generated using Superscript III (Biorad) and fluorescent dyes Cy5 (Amersham).
|
| |
|
| |
| Hybridization protocol |
PolyA, yeast tRNA, bovine rRNA human COT1 DNA was added to combined labeled cDNA samples as a blocker. Samples were added to hybridization buffer (10XSSC, 30% formamide, 0.2% SDS), denatured at 95°C for 3 minutes, and snap cooled on ice. Samples were loaded onto the slide by capillary action under a Corning lifter slip and sealed into a corning hybridization chamber with 30µl water at each as a humidifier. Samples were hybridized to the slides for 18 hours at 55°C in a hybridization oven. Hybridized slides were washed for 10 minutes at room temperature in each of 2XSSC, 0.1%SDS; 1XSSC, 0.1%SDS; 1XSSC; 0.5XSSC; and rinsed for 10 seconds in distilled water.
|
| Scan protocol |
Fluorescence intensity data were collected with a GenePix 4000B scanner (Axon Instruments) with GenePix Pro 4.0 software. Photomultiplier tube gains were set empirically for each channel to optomize intensity and to equalize intensities for control spots on each channel.
|
| Description |
These data represent genes which were expressed in 72 hour phosphate-starved culture versus log phase phosphate-replete cultures.
|
| Data processing |
Collected microarray data were normalized as follows: the sum total measured intensity of all genes from the sample channel (72 hrs) was divided by that of the control channel, and each gene's intensity value from the control channel then was multiplied by this ratio (global normalization). Globally normalized expression values were log2-transformed in order to calculate mean log intensity and log ratios between sample and control values. Local mean normalization was performed using Stata 10 software (StataCorp LP, College Station, TX) across the range of expression levels. The "residuals" from this regression fit (i.e., the distance between observed data and the regression fit,) were used as corrected log ratio values and further analyzed using SAM (Significance Analysis of Microarrays, version 3.01) software to obtain q-values (false discovery rate).
|
| |
|
| Submission date |
Feb 13, 2009 |
| Last update date |
Sep 22, 2009 |
| Contact name |
Petros Karakousis |
| E-mail(s) |
petros@jhmi.edu
|
| Phone |
4105028233
|
| Fax |
4106148173
|
| Organization name |
John Hopkins University school of medicine
|
| Department |
Medicine
|
| Lab |
The center for tuberculosis research
|
| Street address |
1550 Orleans street
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21231 |
| Country |
USA |
| |
|
| Platform ID |
GPL8189 |
| Series (1) |
| GSE14840 |
Phosphate depletion: A novel trigger for Mycobacterium tuberculosis persistence |
|
