|
| Status |
Public on Apr 01, 2010 |
| Title |
Leaves: Champagne non-acclimated 20 days |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
aerial tissues of Champagne non-acclimated 20 days
|
| Organism |
Pisum sativum |
| Characteristics |
variety: Champagne
tissue: leaves (aerial)
|
| Biomaterial provider |
UMR1281 USTL INRA
|
| Treatment protocol |
non-acclimated plants, corresponding to 20 days
|
| Growth protocol |
Plants were grown under greenhouse conditions 19°C day/12°C night, 10h light/day and 300µmol/m²s (phase of nursery). The phase of acclimation was excluded and the phase of nursery was prolonged in order to attempt the same stage of development as acclimated plants (20 days) on the basis of number of degrees x days (Eteve and Derieux, 1982; Ney and Turc, 1993).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using a Tri reagent kit (Euromedex). RNA quantification was done by capillary electrophoresis (Experion, Biorad).
|
| Label |
CY5,CY3
|
| Label protocol |
Twenty-five μg of total RNA were used for each experiment. Reverse transcription and fluorochrome incorporation were performed in a MJ PTC-200 Peltier Thermal Cycler, using oligo-dT (Roche), the Superscript II Kit (Invitrogen) and CY3- and CY5-labelled d-CTP (Amersham). The QIAquick PCR Purification Kit (Qiagen) was used for target purification.
|
| |
|
| Channel 2 |
| Source name |
aerial tissues of Champagne without treatment (d0)
|
| Organism |
Pisum sativum |
| Characteristics |
variety: Champagne
tissue: leaves (aerial)
|
| Biomaterial provider |
UMR1281 USTL INRA
|
| Treatment protocol |
Without temperature treatment (d0)
|
| Growth protocol |
Plants were grown under greenhouse conditions 19°C day/12°C night, 10h light/day and 300µmol/m²s (phase of nursery).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using a Tri reagent kit (Euromedex). RNA quantification was done by capillary electrophoresis (Experion, Biorad).
|
| Label |
CY3,CY5
|
| Label protocol |
Twenty-five μg of total RNA were used for each experiment. Reverse transcription and fluorochrome incorporation were performed in a MJ PTC-200 Peltier Thermal Cycler, using oligo-dT (Roche), the Superscript II Kit (Invitrogen) and CY3- and CY5-labelled d-CTP (Amersham). The QIAquick PCR Purification Kit (Qiagen) was used for target purification.
|
| |
|
| |
| Hybridization protocol |
14-18 h hybridization (DigEasy hybridization buffer; Roche) at 42°C in a Corning chamber. Slides were washed successively in 4x, 2x (with SDS 0.1x), 0.2x, and 0.1x SSC.
|
| Scan protocol |
Images were acquired with a GenePix4000B scanner (Molecular Devices, Sunnyvale, CA, USA)
|
| Description |
Images were analyzed with GenePix Pro 6.0 software (Axon).
|
| Data processing |
Artefactual, saturated, or low-signal spots were eliminated from the analysis and the background subtracted median intensities used for calculations. For each slide a global lowess followed by a print-tip median normalization was performed using R (http://cran.r-project.org/) packages as implemented in Goulphar (Lemoine et al. 2006). In order to exclude possible dye effects, all probes exhibiting an opposite behaviour in the swapped-dye experiment were eliminated from the final analysis. To identify genes displaying a change in expression over repetitions, a script utilizing libraries functions in R with a false discovery rate (FDR) less than 5% was used for all experimental conditions. Only genes with smooth expression profiles were retained. The SAM (Tusher et al. 2001) was used to identify differentially expressed genes over different conditions and log2(ratio) ≥1 and ≤ -1 were used for filtering gene expression profiles. Three biological replicates were used for each analysed condition. Dye labelling for each paired sample was reversed in two subsequent individual hybridizations giving rise to a total of six hybridizations per condition.
|
| |
|
| Submission date |
Nov 25, 2009 |
| Last update date |
Dec 10, 2009 |
| Contact name |
Lucau Anca |
| E-mail(s) |
Anca.Lucau@univ-lille1.fr
|
| Phone |
+33320434428
|
| URL |
http://www.univ-lille1.fr/pdv/labo/microarray.htm
|
| Organization name |
UMR USTL / INRA 1281
|
| Department |
Stress Abiotiques et Différenciation des Végétaux Cultivés
|
| Lab |
Adaptation au froid du pois protéagineux
|
| Street address |
Cité Scientifique SN2/302
|
| City |
Villeneuve d'Ascq |
| ZIP/Postal code |
59000 |
| Country |
France |
| |
|
| Platform ID |
GPL8288 |
| Series (1) |
|
