|
| Status |
Public on May 01, 2008 |
| Title |
Rhodococcus jostii RHA1 incubated at 100% relative humidity for 1 h, replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
mid-exponential phase, benzoate-grown Rhodococcus jostii RHA1 incubated at 100% relative humidity (30°C) for 1 h
|
| Organism |
Rhodococcus jostii RHA1 |
| Characteristics |
mid-exponential phase, benzoate-grown Rhodococcus jostii RHA1 incubated at 100% relative humidity (30°C) for 1 h
|
| Growth protocol |
Grown at 30°C and 200 rpm in W minimal medium amended with 20 mM sodium benzoate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets were vortexed with glass beads, hot phenol, and SDS at final concentrations of 14.3% and 0.9% (vol/vol) respectively. Debris was precipitated with acetate followed by the addition of 4.0 mL phenol-chloroform (1:1, vol/vol). Nucleic acids were precipitated with acetate plus isopropanol, treated with DNase, and purified with an RNeasy mini column (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
Cy-labelled cDNA was prepared by indirect labelling. For two of the three hybridizations, the Cy3/Cy5 dyes were reversed (i.e., cDNA from the T = 1 h treatment was labelled with Cy5 rather than Cy3) to control for dye bias.
|
| |
|
| Channel 2 |
| Source name |
mid-exponential phase, benzoate-grown Rhodococcus jostii RHA1 at time zero (just after filtration onto nitrocellulose membrane)
|
| Organism |
Rhodococcus jostii RHA1 |
| Characteristics |
mid-exponential phase, benzoate-grown Rhodococcus jostii RHA1 at time zero (just after filtration onto nitrocellulose membrane)
|
| Growth protocol |
Grown at 30°C and 200 rpm in W minimal medium amended with 20 mM sodium benzoate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets were vortexed with glass beads, hot phenol, and SDS at final concentrations of 14.3% and 0.9% (vol/vol) respectively. Debris was precipitated with acetate followed by the addition of 4.0 mL phenol-chloroform (1:1, vol/vol). Nucleic acids were precipitated with acetate plus isopropanol, treated with DNase, and purified with an RNeasy mini column (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
Cy-labelled cDNA was prepared by indirect labelling. For two of the three hybridizations, the Cy3/Cy5 dyes were reversed (i.e., cDNA from the T = 1 h treatment was labelled with Cy5 rather than Cy3) to control for dye bias.
|
| |
|
| |
| Hybridization protocol |
The microarray slides were pre-hybridized using 5X SSC containing 0.1% SDS and 0.2% BSA for 45 minutes at 48°C and used immediately for hybridization in a GeneTac HybStation (Genomic Solution). Equal amounts of differentially labelled cDNA – 50 million pixels as measured by ImageQuant 5.2 (Molecular Dynamics) – from T = 1 h and T = 0 cells were hybridized at 42°C for 17 h with agitation using 120 uL per slide of SlideHyb#1 hybridization solution (Ambion). The post-hybridization washing consisted of 3 cycles of 20 second incubations with each of the following solutions: 2X SSC plus 0.1% SDS (medium stringency) at 42°C; 0.1X SSC plus 0.05% SDS (high stringency) at 25°C; and 0.1X SSC (low stringency) at 25°C. Lastly, the slides were dipped in 0.2X SSC and dried by centrifugation at 225 x g for 5 min at room temperature.
|
| Scan protocol |
Microarray slides were scanned with a GenePix 4000B scanner (Axon Instruments) and spot intensities were quantified using Imagene 6.0 (BioDiscovery, Inc.).
|
| Description |
none
|
| Data processing |
Expression ratios were normalized using GeneSpring version 7.2 (Silicon Genetics) by the intensity-dependent Lowess method, with 20% of the data used for smoothing. Replicate spots of the same type were combined to yield a single normalized ratio, which is output for the first ID_REF number representing that spot type.
|
| |
|
| Submission date |
Apr 17, 2008 |
| Last update date |
Apr 20, 2008 |
| Contact name |
Justin Christian LeBlanc |
| Phone |
604-822-5646
|
| Fax |
604-822-6041
|
| Organization name |
University of British Columbia
|
| Department |
Microbiology & Immunology
|
| Lab |
Dr. William W. Mohn
|
| Street address |
2350 Health Sciences Mall
|
| City |
Vancouver |
| State/province |
British Columbia |
| ZIP/Postal code |
V6T 1Z3 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6703 |
| Series (1) |
| GSE10378 |
Desiccation and control transcriptomes of Rhodococcus jostii RHA1 |
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