|
| Status |
Public on Jun 13, 2006 |
| Title |
Treatment 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
E. chrysanthemi exposed to phenolic acids
|
| Organism |
Dickeya chrysanthemi |
| Characteristics |
Gram negative bacterium
Enterobacteriaceae
Plant pathogen
Strain 3937
|
| Biomaterial provider |
Lyon, Fr
|
| Treatment protocol |
Exponentially growing bacteria were treated for 90 min with 0.078 mM each, Salicylic acid, Benzoic acid, t-cinnamic acid for the Treatment.
|
| Growth protocol |
Bacteria were grown at 30C in Luria Bertani medium shaken at 150 rpm for the duration of the experiment (90 min).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using manufacturer's instructions (Qiagen). RNA was quantified using NanoDrop and cDNA synthesized using Superscript (Invitrogen).
|
| Label |
cy3
|
| Label protocol |
cDNA with aminoally-dUTP (Ambion) was labeled with Cy3 or Cy5 and purified on Qiagen MiniElute colums.
|
| |
|
| Channel 2 |
| Source name |
control
|
| Organism |
Dickeya chrysanthemi |
| Characteristics |
Gram negative bacterium
Enterobacteriaceae
Plant pathogen
Strain 3937
|
| Biomaterial provider |
Lyon, Fr
|
| Treatment protocol |
Exponentially growing bacteria were treated for 90 min with 0.078 mM Solvent dimethylsulfoxide (DMSO) as a control.
|
| Growth protocol |
Bacteria were grown at 30C in Luria Bertani medium shaken at 150 rpm for the duration of the experiment (90 min).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using manufacturer's instructions (Qiagen). RNA was quantified using NanoDrop and cDNA synthesized using Superscript (Invitrogen).
|
| Label |
cy5
|
| Label protocol |
cDNA with aminoally-dUTP (Ambion) was labeled with Cy3 or Cy5 and purified on Qiagen MiniElute colums.
|
| |
|
| |
| Hybridization protocol |
Equivalent amounts of cDNA (control and sample) labeled with Cy3 or Cy5 were mixed with 25 µl control target, 105 µl of 2 x hybridization buffer (Agilent Technologies, Inc. CA., USA) and then assembled in a chamber for hybridization. Hybridization was carried out for 17 hours at 60°C in an Agilent oven. After hybridization, the slides were washed at room temperature, and then dried by spin.
|
| Scan protocol |
Arrays were scanned using an ArrayWoRx Auto scanner (Applied Precision Inc) with various exposure settings for Cy3 (595 nm) and Cy5 (685 nm) at 9.756 µm resolution, generating separate TIFF images. Intensity data were acquired from images using SoftWoRx Tracker software.
|
| Description |
Data for specific genes confirmed by Q-RT-PCR.
|
| Data processing |
Images were imported and aligned with probe position (Agilent GAL file) using analyzerDG software automated and manual grid alignment features (MolecularWare Inc). Median signal spot and individual median background intensity values were extracted for each wavelength and imported into GeneSpring (v. 7.2; Agilent), whereby replicate spots in each array were averaged. Background-subtracted signals were corrected for unequal dye incorporation or unequal load of labeled product. The algorithm consisted of a rank consistency filter and normalization using the LOWESS (locally weighted linear regression) method. Saturated spots and the spots shown a reference signal lower than background were excluded from subsequent analyses. Based on standard deviation calculations, probes (genes) having ≥ 1.5 or ≤ 0.66 final sample/control (S/C) ratios were selected as statistically significant sample up-regulated or sample down-regulated. An increase or decrease of average signal log ratio from three biological replicates were evaluated by the GeneSpring software as significantly changed (t-test, P-value ≤ 0.05).
|
| |
|
| Submission date |
Dec 22, 2005 |
| Last update date |
Mar 28, 2006 |
| Contact name |
Michael San Francisco |
| E-mail(s) |
michael.sanfrancisco@ttu.edu
|
| Phone |
806 742-2706
|
| Fax |
806 742-2963
|
| Organization name |
Texas Tech University
|
| Department |
Biological Sciences
|
| Street address |
Flint and Main St
|
| City |
LUBBOCK |
| State/province |
TX |
| ZIP/Postal code |
79409 |
| Country |
USA |
| |
|
| Platform ID |
GPL3280 |
| Series (1) |
|
