|
| Status |
Public on Jan 21, 2010 |
| Title |
Salicyl alcohol vs. control, rep6, dye-swap |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
control cells
|
| Organism |
Populus tremuloides |
| Characteristics |
strain: clone L4
|
| Treatment protocol |
Cell cultures were fed 5 mM salicyl alcohol (treatment) or water (control) 5 days after subculture. Fed and unfed cells were harvested 48 hours after feeding for RNA extraction.
|
| Growth protocol |
Cell cultures derived from Populus tremuloides clone L4 were maintained in WPM media supplemented with 3% sucrose and 2.2 mg/L 2.4-D. Cells were subcultured at 11-day intervals by inoculating 5 mL of cultures to 30 mL of fresh media.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
CTAB extraction
|
| Label |
Cy5
|
| Label protocol |
DNase-treated total RNA and spike mix (Lucidea Universal ScoreCard, Amersham) were reverse transcribed in the presence of aminoallyl-modified dUTP (Sigma) using anchored oligo (dT)20 primer and SuperScript III reverse transcriptase (Invitrogen). Aminoallyl-modified cDNA was purified with the QIAquick PCR purification kit and resuspended in 0.1M sodium carbonate buffer (pH 9.0) for chemical coupling with Cy3 and Cy5 dye (Amersham). The fluorescent-labeled targets were purified with the QIAquick PCR purification kit, and cDNA concentration and Cy dye labeling efficiency measured with a NanoDrop ND-100 spectrophotometer. Aliquots of 50 pmol Cy dye-labeled cDNA were vacuum dried, and stored at -20oC until used.
|
| |
|
| Channel 2 |
| Source name |
salicyl alcohol-fed cells
|
| Organism |
Populus tremuloides |
| Characteristics |
strain: clone L4
|
| Treatment protocol |
Cell cultures were fed 5 mM salicyl alcohol (treatment) or water (control) 5 days after subculture. Fed and unfed cells were harvested 48 hours after feeding for RNA extraction.
|
| Growth protocol |
Cell cultures derived from Populus tremuloides clone L4 were maintained in WPM media supplemented with 3% sucrose and 2.2 mg/L 2.4-D. Cells were subcultured at 11-day intervals by inoculating 5 mL of cultures to 30 mL of fresh media.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
CTAB extraction
|
| Label |
Cy3
|
| Label protocol |
DNase-treated total RNA and spike mix (Lucidea Universal ScoreCard, Amersham) were reverse transcribed in the presence of aminoallyl-modified dUTP (Sigma) using anchored oligo (dT)20 primer and SuperScript III reverse transcriptase (Invitrogen). Aminoallyl-modified cDNA was purified with the QIAquick PCR purification kit and resuspended in 0.1M sodium carbonate buffer (pH 9.0) for chemical coupling with Cy3 and Cy5 dye (Amersham). The fluorescent-labeled targets were purified with the QIAquick PCR purification kit, and cDNA concentration and Cy dye labeling efficiency measured with a NanoDrop ND-100 spectrophotometer. Aliquots of 50 pmol Cy dye-labeled cDNA were vacuum dried, and stored at -20oC until used.
|
| |
|
| |
| Hybridization protocol |
Cy3- and Cy5- labeled cDNA from control and treated samples were suspended in 55 μl of hybridization buffer (50% formamide, 5× SSC, 0.4% SDS and 0.1% BSA). The mixture was denatured at 42oC for 5 min, centrifuged briefly, and applied to the array slide. The slide was covered with a piece of parafilm amd incubated for 36 hours in a hybridization oven (Boekel Scientific) at 40oC with humidity maintained by wet paper towels soaked in the hybridization buffer. The slides were then rinsed three times separately in beakers containing 500 ml of 1× SSC and 0.2% SDS (42oC), followed by 0.1× SSC and 0.2% SDS, and finally in 0.1× SSC before drying the slides using a microarray Air Jet.
|
| Scan protocol |
Arrays were scanned using an Axon Genepix 4000B scanner, and the florescence signal intensity was quantified using the GenePix Pro 5.1 software.
|
| Description |
dye-swap of replicate 3
|
| Data processing |
Data analysis was carried out using GeneSpring 7.3. Replicate spots were averaged and the channels reversed for dye-swap replicates. Data from the two channels were log2 transformed and normalized using the LOWESS algorithm.
|
| |
|
| Submission date |
Oct 01, 2009 |
| Last update date |
Jan 21, 2010 |
| Contact name |
Chung-Jui Tsai |
| E-mail(s) |
cjtsai@uga.edu
|
| Organization name |
University of Georgia
|
| Department |
School of Forestry and Natural Resources
|
| Street address |
120 Green Street
|
| City |
Athens |
| State/province |
GA |
| ZIP/Postal code |
30606 |
| Country |
USA |
| |
|
| Platform ID |
GPL9334 |
| Series (1) |
| GSE18360 |
Gene expression response of Populus tremuloides cell suspension cultures to salicyl alcohol feeding |
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