|
| Status |
Public on Jun 10, 2010 |
| Title |
Rhododoccus jostii RHA1 cells were grown on membrane filter placed on W minimal solid medium with biphenyl, replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
solid-support dependent genes, Rhododoccus jostii RHA1 cells were grown on membrane filter placed on W minimal 2%-agar solid medium with vapor of biphenyl, replicate 2
|
| Organism |
Rhodococcus jostii RHA1 |
| Characteristics |
strain: Rhododoccus jostii RHA1
substrate: biphenyl
medium: solid medium
|
| Biomaterial provider |
none
|
| Treatment protocol |
none
|
| Growth protocol |
Rhodococcus jostii RHA1 cells were grown on membrane filter placed on W minimal 2%-agar solid medium with vapor of biphenyl at 30°C
|
| 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. the Cy3/Cy5 dyes were reversed to control for dye bias.
|
| |
|
| Channel 2 |
| Source name |
mid-exponential phase, Rhodococcus jostii RHA1 was grown in W minimal liquid medium with biphenyl
|
| Organism |
Rhodococcus jostii RHA1 |
| Characteristics |
strain: Rhodococcus jostii RHA1
substrate: biphenyl
medium: liquid medium
|
| Biomaterial provider |
none
|
| Treatment protocol |
none
|
| Growth protocol |
mid-exponential phase, Rhodococcus jostii RHA1 was grown in W minimal liquid medium with 10 mM biphenyl at 30°C and 120 rpm
|
| 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. the Cy3/Cy5 dyes were reversed 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 = 48 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 6.1 (BioDiscovery, Inc.).
|
| Description |
none
|
| Data processing |
Expression ratios were normalized using GeneSpring version 7.3.1 (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.
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| |
|
| Submission date |
Jun 08, 2010 |
| Last update date |
Jun 09, 2010 |
| Contact name |
Toju Iino |
| E-mail(s) |
iino@stn.nagaokaut.ac.jp
|
| Phone |
+81-258-47-9427
|
| Fax |
+81-258-47-9450
|
| Organization name |
Nagaoka University of Technology
|
| Department |
Dept. Bioengineering
|
| Lab |
Microbial Engineering
|
| Street address |
1603-1 Kamitomioka
|
| City |
Nagaoka |
| State/province |
Niigata |
| ZIP/Postal code |
940-2188 |
| Country |
Japan |
| |
|
| Platform ID |
GPL7627 |
| Series (1) |
| GSE22239 |
solid-support dependent genes, Rhododoccus jostii RHA1 cells were grown on membrane filter placed on W minimal 2%-agar solid medium with vapor of biphenyl |
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