|
| Status |
Public on Aug 26, 2009 |
| Title |
DelCu60T24_Rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Populus deltoides roots from plants grown in basal nutrient solution supplemented with copper 60 µM, after 24 hours of incubation (Cu60, T24)
|
| Organism |
Populus deltoides |
| Characteristics |
Plants of a Populus deltoides clone grown in a hydroponic system during 4 weeks were incubated in basal nutrient solution (Hoagland’s modiefied salt, ¼ strength) supplemented with copper 60 µM and maintained during 24 h before root harvest.
|
| Biomaterial provider |
Centro Tecnológico del Álamo (Universidad de Talca, Chile)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Roots were ground in liquid nitrogen, bead beat in QIAgen buffer RLC and then RNA were isolated with QIAgen RNeasy Plant kit
|
| Label |
[33P]dCTP, [33P]dATP
|
| Label protocol |
Total RNA were treated with DNase and cDNA were aplified with SMART-PCR cDNA Synthesis Kit (BD BioSciences). cDNA probes were labelled with 30 μCi [33P]dCTP and 30 μCi [33P]dATP using the Prime-a-Gene Kit (Promega). The unincorporated labeled nucleotides were removed using QIAquick columns (QIAGEN)
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| |
|
| Hybridization protocol |
Nylon arrays were preincubated in 30 ml of a hybridization solution (5XSSC, 5XDenhardt’s solution, 0.5% SDS, 50µg/ml shared salmon sperm DNA) for 4 h at 65°C in a rotating hybridization incubator. The arrays were then incubated in 10 ml of fresh hybridization solution containing the 33P-labeled probe at 65°C for 22 h. The hybridized arrays were washed successively for 3 x 5 min in 2X SSC at room temperature; 2 x 20 min in 2X SSC, 0.05% SDS (65°C); 2 X 20 min in 1X SSC, 0.1% SDS (65°C); and 2 x 20 min in 0.1X SSC, 0.1% SDS (65°C).
|
| Scan protocol |
Arrays were exposed to phosphorimaging screen (Eastman Kodak Company) and cDNA spots intensities were visualized by scanning at a resolution of 50 µm per pixel in a Personal Molecular Imager FX (Bio-Rad).
|
| Description |
Spots positions in the 5x5 grid within the 384 blocks generated by the arrayer were retrieved in scanned 16-bits TIFF images by using the XDotsReader spot finder software (COSE, Paris France). In the raw file generated by XDotsReader, the spot position (1 to 23, odd numbers) is followed by block position (A to P; 1 to 24) in the 'def.coord' column and the raw signal intensity is given in the 'sel' column
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| Data processing |
Data were processed as follow: the mean value of all empty spots positions was calculated and then a flag-limit corresponding to 2 x this value was set; all spots that showed raw intensities below this flag-limit in all treatments were removed from the analysis; remaining spots intensities were centered-normalized to mean intensity value within each experiment. The statistical analysis was implemented using the MIXED procedure of the SAS system utilizing an approach based on linear mixed models
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| |
|
| Submission date |
Dec 02, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Fernando Guerra |
| E-mail(s) |
[email protected]
|
| Phone |
56-71-200281
|
| Organization name |
Universidad de Talca
|
| Department |
Instituto de Biologia Vegetal y Biotecnologia
|
| Street address |
2 Norte 685
|
| City |
Talca |
| State/province |
Talca |
| ZIP/Postal code |
0000000 |
| Country |
Chile |
| |
|
| Platform ID |
GPL4887 |
| Series (1) |
| GSE9748 |
Identification of differentially regulated genes in Populus deltoides subjected to copper stress using 4.6K cDNA arrays |
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