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Status |
Public on Jun 10, 2010 |
Title |
Rhodocuccus jostii RHA1 was grown in the sterilized soil with biphenyl, replicate 3 |
Sample type |
RNA |
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Channel 1 |
Source name |
Rhodocuccus jostii RHA1 was grown in the sterilized soil with biphenyl at 3 day
|
Organism |
Rhodococcus jostii RHA1 |
Characteristics |
strain: Rhodocuccus jostii RHA1 growth conditions: grown in the sterilized soil with biphenyl at 3 day
|
Biomaterial provider |
none
|
Treatment protocol |
none
|
Growth protocol |
Rhodococcus jostii RHA1 was grown in the sterilized soil with 10 mM 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.
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|
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Channel 2 |
Source name |
Rhodococcus jostii RHA1 was grown in W minimal liquid medium with biphenyl
|
Organism |
Rhodococcus jostii RHA1 |
Characteristics |
strain: Rhodococcus jostii RHA1 growth conditions: grown in W minimal liquid medium with biphenyl
|
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
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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) |
GSE22240 |
Rhododoccus jostii RHA1 was grown in the sterilized soil with biphenyl |
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