|
| Status |
Public on Jul 08, 2013 |
| Title |
WT Rep 4 vs Ph7 Rsh1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Brucella liquid culture (minimal medium)
|
| Organism |
Brucella suis 1330 |
| Characteristics |
genotype/variation: Wild-type
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Rneasy (Qiagen) following manufacturer's instructions
|
| Label |
Cy5 with Uref on Cy3
|
| Label protocol |
Total RNA (30 µg) was first converted to cDNAs incorporating a chemically reactive nucleotide analog (amino allyl-dUTP) using Superscript III reverse transcriptase and random hexamers (Invitrogen). This cDNA was then post labeled with the reactive forms of fluoroLink Cy3-NHS esters (Amersham), which bind to the modified nucleotides. The labeled cDNAs were then combined with 0.5 µg of labeled gDNA to a final volume of 35 μL.
|
| |
|
| Channel 2 |
| Source name |
Brucella liquid culture (minimal medium)
|
| Organism |
Brucella suis 1330 |
| Characteristics |
genotype/variaton: rsh mutant
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Rneasy (Qiagen) following manufacturer's instructions
|
| Label |
Cy5 with Uref on Cy3
|
| Label protocol |
Total RNA (30 µg) was first converted to cDNAs incorporating a chemically reactive nucleotide analog (amino allyl-dUTP) using Superscript III reverse transcriptase and random hexamers (Invitrogen). This cDNA was then post labeled with the reactive forms of fluoroLink Cy3-NHS esters (Amersham), which bind to the modified nucleotides. The labeled cDNAs were then combined with 0.5 µg of labeled gDNA to a final volume of 35 μL.
|
| |
|
| |
| Hybridization protocol |
Samples were heated at 95°C for 5 min and then kept at 45 °C until hybridization, when 35 μL of 2× formamide-based hybridization buffer (50% formamide; 10× SSC; 0.2% SDS) was added to each sample. Samples were then well-mixed and applied to custom 3.2 K Brucella oligo-arrays. Prior to hybridization, oligo-arrays were pretreated by washing in 0.2% SDS, followed by 3 washes in distilled water, and immersed in pre-hybridization buffer (5 × SSC, 0.1% SDS; 1% BSA in 100 ml of water) at 45°C for at least 45 min. Immediately before hybridization, the slides were washed 4 × in distilled water, dipped in 100% isopropanol for 10 sec and dried by centrifugation at 1,000 × g for 2 min. Four slides for each condition (Wild-type and rsh mutant) were hybridized at 45°C for ~ 20 h in a dark, humid chamber (Corning) and then washed for 10 min at 45°C with low stringency buffer (1× SSC, 0.2% SDS), followed by two 5-min washes in a higher stringency buffer (0.1 × SSC, 0.2% SDS and 0.1 × SSC) at room temperature with agitation.
|
| Scan protocol |
Hybridized microarrays were scanned using a GenePix 4000A dual-channel (635 nm and 532 nm) confocal laser scanner (Axon Instruments). The genes represented on the arrays were adjusted for background and normalized to internal controls using image analysis software (GenePixPro 4.0; Axon Instruments Inc.).
|
| Description |
Wildtype versus Rsh for pH7 condition
H7-WT2-2009-02-10:URefMerged:H8-Rsh2-2009-02-10
Virtual dual channel design; Channel 1 (Cy3) on each hybridization was a universal reference (Uref) used as a reference point to merge two (2 channel arrays) into a virtual 2 channel array with wild-type versus rsh mutant.
|
| Data processing |
Global scaling to 200 across all aways, LOWESS normalized, background subtracted data. Channel 1 (Cy3) was a universal reference (Uref) used as a reference point to merge two (2 channel arrays) into a virtual 2 channel array with wild-type versus rsh mutant. Non hybridized probes labeled as “empty” in the raw data files where excluded from the analysis.
|
| |
|
| Submission date |
Feb 26, 2013 |
| Last update date |
Jul 08, 2013 |
| Contact name |
Stephan Kohler |
| E-mail(s) |
stephan.kohler@cpbs.cnrs.fr
|
| Organization name |
CNRS
|
| Lab |
UMR 5236
|
| Street address |
1919 Route de Mende
|
| City |
Montpellier |
| ZIP/Postal code |
34095 |
| Country |
France |
| |
|
| Platform ID |
GPL7612 |
| Series (1) |
| GSE44688 |
Global Rsh-dependent transcription profile of Brucella suis during stringent response unravels adaptation to nutrient starvation and cross-talk with other stress responses. |
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