|
| Status |
Public on Mar 15, 2007 |
| Title |
Bpre_C12Rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Beaupré leaves inoculated with Melampsora larici-populina 98AG31 virulent strain, 12 hours post-inoculation (Compatible, C12)
|
| Organism |
Populus trichocarpa x Populus deltoides |
| Characteristics |
Detached leaves of Populus trichocarpa x Populus deltoides 'Beaupré' cuttings grown in greenhouses were inoculated with Melampsora larici-populina 98AG31 virulent strain (100000 spores/ml), maintained in vitro under constant illumination at 24°C and harvested 12 hours post-inoculation
|
| Biomaterial provider |
Institut National de la Recherche Agronomique (INRA Nancy, France)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Leaves were ground in liquid nitrogen, bead beat in QIAgen buffer RLT and then RNA were isolated with QIAgen RNeasy 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. Statistical analyses between replicates were further processed with the CyberT statistical framework (http://visitor.ics.uci.edu/genex/cybert/index.shtml)
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| |
|
| Submission date |
Feb 23, 2007 |
| Last update date |
Feb 26, 2007 |
| Contact name |
Sebastien Duplessis |
| Organization name |
INRA
|
| Department |
UMR 1136 IAM
|
| Lab |
Ecogenomics of interactions
|
| Street address |
INRA Centre Grand Est - Nancy
|
| City |
Champenoux |
| ZIP/Postal code |
54280 |
| Country |
France |
| |
|
| Platform ID |
GPL4887 |
| Series (1) |
| GSE7121 |
Identification of rust-induced genes in poplar using Populus 4.6K cDNA macroarray |
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