|
| Status |
Public on Feb 01, 2017 |
| Title |
549628A03 - FEWSB4_4 vs FEWWA4_4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
FEWWA4_4
|
| Organism |
Pisum sativum |
| Characteristics |
genotype: mutant (CAMEOR)
tissue: leaf
|
| Treatment protocol |
Name:Well Watered - environmental treatment - abiotic stress,water-holding capacity of the substrate:quantity 100percent . Pea seeds were sown in 2L pots filled with a mixture (3:1, vol/vol) of perlite and sand and were inocculated with Rhizobium. Watering with a nutritve solution without nitrate (to allow nodulation) was unlimited (drainage conditions) until flowering. At flowering, plants were weighted three times a day in order to maintain a soil relative water content correspondong to the maximum (100%) water holding capacity of the substrate for all the plants. Twelve days after flowering irrigation was stopped for half of the plants (WS plants) until soil water relative content reached 50% of the maximum water-holding capaciy of the substrate. Once this target value was reached (24 hours), it was kept to 50% until samples harvest.
|
| Growth protocol |
caulin leaf - Growth conditions (1-2-3-4):1. Media: perlite:sand (vol:vol 3:1) - hygrometry: 50% - Temperature: 15°C night; 18°C day - Light: 16h photoper
|
| Extracted molecule |
total RNA |
| Extraction protocol |
FEWWA4_4:150mg. (DNeasy_Plant_Qiagen.pdf)
|
| Label |
Cy5
|
| Label protocol |
labelling Cy3 and Cy5 direct, amplification=yes, aRNA 5 ug. (Labelling_protocol.txt) Labelling protocol: 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).
|
| |
|
| Channel 2 |
| Source name |
FEWSB4_4
|
| Organism |
Pisum sativum |
| Characteristics |
genotype: mutant (CAMEOR)
tissue: leaf
|
| Treatment protocol |
Name:Water stressed - environmental treatment - abiotic stress,water-holding capacity of the substrate:quantity 50percent .
|
| Growth protocol |
caulin leaf - Growth conditions (1-2-3-4):1. Media: perlite:sand (vol:vol 3:1) - hygrometry: 50% - Temperature: 15°C night; 18°C day - Light: 16h photoper
|
| Extracted molecule |
total RNA |
| Extraction protocol |
FEWSB4_4:150mg. (DNeasy_Plant_Qiagen.pdf)
|
| Label |
Cy3
|
| Label protocol |
labelling Cy3 and Cy5 direct, amplification=yes, aRNA 5 ug. (Labelling_protocol.txt) Labelling protocol: 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).
|
| |
|
| |
| Hybridization protocol |
FEWWA4_4 Cy5 / FEWSB4_4 Cy3 : 30pmol. (Hyb_Scan_Nimb_GEO.doc)
|
| Scan protocol |
Mapix, Cy3:pmt voltage 532nm,480V,laser power 35%, Cy5:635nm,pmt voltage 480V,laser power 30%
|
| Description |
harvest date:27-01-13
harvest date:03-02-13
What is the effect of a moderate water stress on seed filling (reserve accumulation) and nitrogen remobilisation in pea (Pisum sativum)
|
| Data processing |
For each array, the raw data comprised the logarithm of median feature pixel intensity at wavelengths Cy5 (red) and Cy3 (green).For each array, a global intensity-dependent normalization using the loess procedure (Yang et al., 2002) was performed to correct the dye bias.Log-ratios are then averaged over the duplicate probes to get a value per gene.
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| |
|
| Submission date |
Jan 13, 2017 |
| Last update date |
Feb 01, 2017 |
| Contact name |
Stéphanie Pateyron |
| E-mail(s) |
pateyron@evry.inra.fr
|
| Organization name |
IPS2_Institute of Plant Sciences Paris-Saclay
|
| Lab |
Transcriptomic Plateforme POPS
|
| Street address |
Rue de Noetzlin _ Batiment 630
|
| City |
Orsay |
| ZIP/Postal code |
91405 |
| Country |
France |
| |
|
| Platform ID |
GPL17462 |
| Series (1) |
| GSE93630 |
12plex_pea_2013_02_f-Water stress and seed filling in pea |
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