|
| Status |
Public on Jul 16, 2010 |
| Title |
vtrB replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
wild type
|
| Organism |
Vibrio parahaemolyticus |
| Characteristics |
strain: RIMD 2210633
|
| Growth protocol |
Cells were grown to the OD600=1.0 at 37˚C in Luria-Bertani containing medium 0.5% NaCl.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified using Trizol (Invitrogen) and digested DNA with TURBO DNA-free (Ambion), then carryed out RNeasy mini Kit (Qiagen) according to the mnufacture's instruction.
|
| Label |
Cy5
|
| Label protocol |
First, 20 µg of total RNA were primed with 2 µl of 5 µg/µl random hexamer at 70˚C for 5 min then reversed transcribed at 46˚C for 2 h in the presence of 800 U SuperScript III RTase (Invitrogen), and 5 mM each dATP, dTTP, dCTP, with 2 mM dTTP, 3 mM aminoallyl-modified dUTP (Sigma). The aminoallyl-labeled cDNA was purified by phenol-chloroform extraction and ethanol precipitation. Precipitated cDNA was dried and resuspended in 10 µl of 50 mM NaHCO3. After adding 10 µl of dimethyl sulfoxide solution containing Cy3 or Cy5 mnonofunctional reactive dye (GE healthcare), the sample was incubated at room temperature in the dark for 1h to allow the dye to couple with DNA. the fluorescently labeled DNA was finally purified by the CentriCep spin column (Princeton Sepatations Inc.).
|
| |
|
| Channel 2 |
| Source name |
vtrB
|
| Organism |
Vibrio parahaemolyticus |
| Characteristics |
strain: RIMD 2210633 vtrB mutant
|
| Growth protocol |
Cells were grown to the OD600=1.0 at 37˚C in Luria-Bertani containing medium 0.5% NaCl.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified using Trizol (Invitrogen) and digested DNA with TURBO DNA-free (Ambion), then carryed out RNeasy mini Kit (Qiagen) according to the mnufacture's instruction.
|
| Label |
Cy3
|
| Label protocol |
First, 20 µg of total RNA were primed with 2 µl of 5 µg/µl random hexamer at 70˚C for 5 min then reversed transcribed at 46˚C for 2 h in the presence of 800 U SuperScript III RTase (Invitrogen), and 5 mM each dATP, dTTP, dCTP, with 2 mM dTTP, 3 mM aminoallyl-modified dUTP (Sigma). The aminoallyl-labeled cDNA was purified by phenol-chloroform extraction and ethanol precipitation. Precipitated cDNA was dried and resuspended in 10 µl of 50 mM NaHCO3. After adding 10 µl of dimethyl sulfoxide solution containing Cy3 or Cy5 mnonofunctional reactive dye (GE healthcare), the sample was incubated at room temperature in the dark for 1h to allow the dye to couple with DNA. the fluorescently labeled DNA was finally purified by the CentriCep spin column (Princeton Sepatations Inc.).
|
| |
|
| |
| Hybridization protocol |
Cy3- or Cy5-labeled probes (apploximately 20 µl for each) were mixed, and 3 µl of 10% SDS, 6 µl of 1 mg/ml human Cot-1 DNA (Invitrogen) and 15 µl of 20X SSC (3 M NaCl, 0.3 M trisodium citrate 2H2O, pH 7.0) were added to the mixture. After incubation at 96˚C for 2 min, the denatured sample was applied to a microarray slide and incubated on an MAUI hybridization chamber at 60˚C for 16h. The slide was then washed twice in 2X SSC/0.1% SDS solution at 60˚C for 10 min, twice in 0.2X SSC/0.1% SDS solution at room temperature for 10 min, and twice in 0.2X SSC solution at room temperature for another 10 min. Finally, the slide was briefly rinsed with ethanol and dried by centrifugation.
|
| Scan protocol |
Scanned with a ScanArrayExpress scanner (PerkinElmer). Obtained data were analyzed by the ScanArrayExpress software ver. 3.0.
|
| Description |
none
|
| Data processing |
LOWESS normalaized, background subtracted data obtained from log2 of processed Cy3 signal/processed Cy5 signal.
|
| |
|
| Submission date |
Jul 21, 2009 |
| Last update date |
Jul 21, 2009 |
| Contact name |
Toshio Kodama |
| E-mail(s) |
kodama@biken.osaka-u.ac.jp
|
| Organization name |
OSAKA university
|
| Street address |
3-1 Yamadaoka
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
565-0871 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6058 |
| Series (1) |
| GSE17242 |
RIMD 2210633 wild type versus vtrA or vtrB mutants |
|
