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| Status |
Public on Mar 10, 2015 |
| Title |
0.25mic-90min-repeat 3 |
| Sample type |
RNA |
| |
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| Channel 1 |
| Source name |
0.25mic-90min
|
| Organism |
Shigella flexneri |
| Characteristics |
Overnight cultures were diluted in freshly prepared cation-adjusted MHB to optical density at 600 nm (OD600) of about 0.05.Then the diluted cultures were allowed to continue growing at 37°C with shaking (200 rpm).When they were grown to early exponential growth phase (an OD600 of about 0.3), furazolidone was added from the 400×stock dissolved in DMSO into the cultures to give final concentrations of 0.25× MIC. At 90 min after treatment, samples were collected and washed twice with PBS for subsequent RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by using SV RNA Isolation System .Totle RNA was extracted according to the manufacturer’s instructions.
|
| Label |
cy5
|
| Label protocol |
According to the manufacturer’s instructions, the cDNA was labeled with incorporation of fluorescent dyes (Amersham) except that a different dNTP mix was used (10× mix: 1.2 mM each dATP, dGTP, and dTTP;0.6 mM dCTP;10 mM Tris, pH 8.0;1 mM EDTA). Cy5-dCTP was used.
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| |
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| Channel 2 |
| Source name |
control-90min
|
| Organism |
Shigella flexneri |
| Characteristics |
Overnight cultures were diluted in freshly prepared cation-adjusted MHB to optical density at 600 nm (OD600) of about 0.05.Then the diluted cultures were allowed to continue growing at 37°C with shaking (200 rpm).When they were grown to early exponential growth phase (an OD600 of about 0.3), DMSO without the antimicrobial was added to control cultures at a final concentration of 0.25% (vol/vol) .At 90 min after treatment, samples were collected and washed twice with PBS for subsequent RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by using SV RNA Isolation System .Totle RNA was extracted according to the manufacturer’s instructions.
|
| Label |
cy3
|
| Label protocol |
According to the manufacturer’s instructions, the cDNA was labeled with incorporation of fluorescent dyes (Amersham) except that a different dNTP mix was used (10× mix: 1.2 mM each dATP, dGTP, and dTTP;0.6 mM dCTP;10 mM Tris, pH 8.0;1 mM EDTA). Cy3-dCTP was used.
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| |
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| |
| Hybridization protocol |
Prior to hybridization, the labeled cDNA was purified with QIAquick PCR purification kit (Qiagen), and then each pair of labeled cDNA: one is drug treated sample and the other is corresponding control sample, were mixed and dried by vacuum centrigugation. 1. Mark positions of array grids using a diamond tipped pen. Bake slides for 2-4h at 80°C or UV crosslink twice using the autocrosslink program of a Stratalinker (Stratagene) Slides are then blocked using the method of Diehl et al in Nucleic Acids Research, 2001, Vol 29, No.7 e38 except slides were incubated in water at 95°C instead of boiling for 2 minutes, then rinsed in 95% ethanol for 1 minute and centrifuged dry at 1200 rpm for 5 minutes at room temp, preferably in an enclosed slide chamber (with bottom lined with blotting paper), otherwise ensure the centrifuge bowl and rotor are as dust free as possible. Place the slides in a 5-position plastic slide cassette or slide chamber until ready for use.
2. To 10 µl of dried down labelling reactions add 1.5 µl of 50 X Denhardts solution, 2.25 µl of 20 X SSC, 1.125 µl of E. coli tRNA (10 µg/µl) and 0.375 µl of 1M HEPES, pH7.0. Add 0.375 µl of 10% SDS to the mixture. The hyb components (except SDS) can be mixed together as a stock solution and added to labelling reactions.
3. Incubate 100 °C X 2 min. Let stand on the bench for 5 - 10 min. Do not place on ice or the SDS will precipitate.
4. Spin in a microfuge at full speed for 5 min, transfer to clean tube and repeat spin
5. Place array slide in the hyb chamber.
6. Pipette hyb. solution towards one edge of an array.
7. Place edge of a clean coverslip on the edge of the array.
8. Position fine nosed forceps under the coverslip.
9. Using the forceps slowly lower the coverslip down onto the hyb. solution and the array.
10. Apply 20 µl of 3 X SSC around the 4 corners of the slide using a pipette (approximately 5 µl per corner). This will maintain the correct humidity in the hybridisation chamber.
11. Place the chambers at the bottom of a water bath at 63°C overnight.
12. Place the slides directly into the heated wash solution, 2 X SSC, 0.1% SDS at 63°C and agitate to remove the cover slips (5 mins.). We use a thermocouple attached to a hot-plate stirrer for this step. Repeat.
13. Transfer slide rack to a solution of 1 X SSC at room temp. and agitate for 5 min. Repeat.
14. Wash in 0.2 X SSC for 5 min. Repeat.
15. Spin dry 1200 rpm for 5 min at room temp in an enclosed slide chamber (with blotting paper lining the bottom of the chamber).
16. Scan.
|
| Scan protocol |
The slides were scanned by using a GenePix 4000B scanner (Axon Instruments). The lasers of the scanner produce wavelengths of 532 nm for Cy3- and 635 nm for Cy5-labeled cDNA. Fluorescent spots and local background intensities were quantified using GenePix Pro 6.0 software (Axon).Signal intensities were corrected by subtracting the local background value.
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| Description |
S. flexneri 2a strain 301 (Sf301) was used in our study , The germ was routinely grown at 37°C with shaking (200 rpm) on cation-adjusted MUELLER-HINTON broth (caMHB) containing 20 mg of calcium and 10 mg of magnesium per liter. Furazolidone obtained from Sigma Company was resolved in dimethyl sulfoxide (DMSO) diluted with caMHB to final concentrations. Minimal inhibitory concentration of Furazolidone for Sf301 was determined by using the broth microdilution technique according to NCCLS guidelines
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| Data processing |
the data set were filtered so that spots designated as Not Found flagged features, as well as Bad and Empty features were excluded from further analysis. Moreover, spots either with signal-to-noise ratio below the detection limit of 3 or containing less than 55 percents of pixels which were two standard deviations above background in both channels were discarded from the dataset. After filtration, Lowess normalizations were performed with the fluorescent signal ratios (Cy5/Cy3) by using Tiger MIDAS V2.19 software (Tiger). VALUEs indicates the log2 ratio of cy5/Cy3 of genes.
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| Submission date |
Apr 09, 2007 |
| Last update date |
Mar 10, 2015 |
| Contact name |
Hua Fu |
| Organization name |
Institute of pathogen biology
|
| Street address |
6#, Rongjing East Street
|
| City |
Beijing |
| ZIP/Postal code |
100176 |
| Country |
China |
| |
|
| Platform ID |
GPL5068 |
| Series (1) |
| GSE7478 |
cDNA-Shigella flexnerri- Furazolidone |
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