|
| Status |
Public on Nov 20, 2008 |
| Title |
V. parahaemolyticus cDNA 3%LB early logarithmic growth at 37°C vs cDNA 3%LB late logarithmic/early stationary growth |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
cDNA derived from cells grown at 37°C in LB 3% NaCl harvested at OD600=3.0
|
| Organism |
Vibrio parahaemolyticus RIMD 2210633 |
| Characteristics |
Strain: RIMD2210633
|
| Growth protocol |
To obtain non-replicating bacteria, cells were spread onto Luria-Bertani (LB) Agar followed by a 24 h incubation t 20ºC and a 24 h incubation at 4ºC. Other samples were grown at 37ºC in liquid LB with 3% NaCl or liquid M9 with 3% NaCl and 0.4% glucose until an OD600 of 0.2, 0.5 or 3.0 was reached.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Genomic DNA was purified using the DNeasy Tissue Kit (Qiagen) according to the manufacturer's instruction. RNA was extracted using TRIzol Reagent (Invitrogen) and purified with RNeasy Mini Kit (Qiagen) following the manufacturers protocols.
|
| Label |
Cy5
|
| Label protocol |
For generation of aminoallyl-labelled product from gDNA, the general procedures accompanying BioPrime Array CGH Genomic Labelling System (Invitrogen) were followed with a few modifications. Genomic DNA (2 μg) was incubated for 5 min at 95ºC with 20 μl 2.5X random primer solution in 44 μl reactions. Mixtures were cooled on ice for 3 min before addition of 5 μl 10X dNTP-aminoallyl dUTP (5 mM dATP, dCTP and dGTP, 2 mM dTTP and 3 mM aadUTP, Sigma) and 1 μl Exo-Klenow polymerase (40U, Invitrogen) and a further incubation at 37ºC for 1 h. Reactions were cleaned, precipitated and washed. For generation of aminoallyl-labelled product from total RNA, reagents included in SuperScript™ III Reverse Transcriptase Kit (Invitrogen) were used. In brief, 20 μg RNA was mixed with 10 μg random hexamers in a 22 μl reaction that was incubated at 70ºC for 5 min before cooling on ice. Addition of 5X First-Strand Buffer (8 μl), 0.1 M DTT (2 μl), SuperScript™ III RT (4 μl) and 10X dNTP-aminoallyl dUTP (4 μl) was followed by a 3 h incubation at 46ºC. Samples were cleaned, precipitated and washed. Finally, aminoallyl-labelled nucleic acids were resuspended in 10 μl 50 mM NaCO3 (pH 9.0), and 10 μl of either Cy3 or Cy5 monofunctional reactive dye (GE Healthcare) was added. Mixtures were incubated in the dark for 1 h at room temperature and unincorporated dye was removed on CENTRI-SEP Columns (Princeton Separations) following the manufacturer’s instructions before precipitation and wash.
|
| |
|
| Channel 2 |
| Source name |
cDNA derived from cells grown at 37°C in LB 3% NaCl harvested at OD600=0.2
|
| Organism |
Vibrio parahaemolyticus RIMD 2210633 |
| Characteristics |
Strain: RIMD2210633
|
| Growth protocol |
To obtain non-replicating bacteria, cells were spread onto Luria-Bertani (LB) Agar followed by a 24 h incubation t 20ºC and a 24 h incubation at 4ºC. Other samples were grown at 37ºC in liquid LB with 3% NaCl or liquid M9 with 3% NaCl and 0.4% glucose until an OD600 of 0.2, 0.5 or 3.0 was reached.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Genomic DNA was purified using the DNeasy Tissue Kit (Qiagen) according to the manufacturer's instruction. RNA was extracted using TRIzol Reagent (Invitrogen) and purified with RNeasy Mini Kit (Qiagen) following the manufacturers protocols.
|
| Label |
Cy3
|
| Label protocol |
For generation of aminoallyl-labelled product from gDNA, the general procedures accompanying BioPrime Array CGH Genomic Labelling System (Invitrogen) were followed with a few modifications. Genomic DNA (2 μg) was incubated for 5 min at 95ºC with 20 μl 2.5X random primer solution in 44 μl reactions. Mixtures were cooled on ice for 3 min before addition of 5 μl 10X dNTP-aminoallyl dUTP (5 mM dATP, dCTP and dGTP, 2 mM dTTP and 3 mM aadUTP, Sigma) and 1 μl Exo-Klenow polymerase (40U, Invitrogen) and a further incubation at 37ºC for 1 h. Reactions were cleaned, precipitated and washed. For generation of aminoallyl-labelled product from total RNA, reagents included in SuperScript™ III Reverse Transcriptase Kit (Invitrogen) were used. In brief, 20 μg RNA was mixed with 10 μg random hexamers in a 22 μl reaction that was incubated at 70ºC for 5 min before cooling on ice. Addition of 5X First-Strand Buffer (8 μl), 0.1 M DTT (2 μl), SuperScript™ III RT (4 μl) and 10X dNTP-aminoallyl dUTP (4 μl) was followed by a 3 h incubation at 46ºC. Samples were cleaned, precipitated and washed. Finally, aminoallyl-labelled nucleic acids were resuspended in 10 μl 50 mM NaCO3 (pH 9.0), and 10 μl of either Cy3 or Cy5 monofunctional reactive dye (GE Healthcare) was added. Mixtures were incubated in the dark for 1 h at room temperature and unincorporated dye was removed on CENTRI-SEP Columns (Princeton Separations) following the manufacturer’s instructions before precipitation and wash.
|
| |
|
| |
| Hybridization protocol |
Cy3- or Cy5- labeled probes (21.6 µl each) were mixed and 18 μl 20X SSC (3 M NaCl, 0.3 M HOC(COONa)(CH2COONa)2), 7.2 μl human CotI DNA (Invitrogen) and 3.6 μl 10% SDS was added. Hybridisation mixtures were incubated at 96ºC for 5 min, chilled on ice and kept at 55ºC until application onto microarray slides. Slides were covered with a MAUI AO Lid and insertion into MAUI hybridization chambers (BioMicro Systems) before application of the pre-warmed hybridisation mixture and 16 h incubation at 55ºC followed. After hybridisation, slides were washed twice in 2X SSC, 0.1 % SDS at 55ºC for 10 min, twice in 0.2X SSC, 0.1 % SDS at room temperature for 10 min, twice in 0.2X SSC at room temperature for 10 min and finally in ethanol for 1 min.
|
| Scan protocol |
Slides were scanned with a ScanArrayExpress scanner (PerkinElmer). Obtained data was analysed by ScanArrayExpress software version 3.0 (PerkinElmer).
|
| Description |
none
|
| Data processing |
LOWESS normalised, background subtracted data obtained from log2 of processed Cy3 signal/processed Cy5 signal.
|
| |
|
| Submission date |
Dec 19, 2007 |
| Last update date |
Nov 19, 2008 |
| Contact name |
Kaori Izutsu |
| Organization name |
Osaka University
|
| Department |
Research Institute for Microbial Diseases
|
| Lab |
Laboratory of Genomic Research on Pathogenic Bacteria
|
| Street address |
1-1 Yamadaoka
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
565-0871 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6058 |
| Series (1) |
| GSE9968 |
Examination of growth mode dependent replication dynamics and expression levels for Vibrio parahaemolyticus RIMD2210633. |
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