sample type: Pisum sativum L - harvest date:27-01-14
Treatment protocol
Name:GRWW - 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
seed - 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 photoperiod
Extracted molecule
total RNA
Extraction protocol
GRWWA_4:150mg.
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).
sample type: Pisum sativum L - harvest date:03-02-14
Treatment protocol
Name:GRWW - 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
seed - 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 photoperiod
Extracted molecule
total RNA
Extraction protocol
GRWWB_4:150mg.
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
GRWWA_4 Cy5 / GRWWB_4 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.
Scan protocol
Mapix, Cy3:pmt voltage 532nm,480V,laser power 35%, Cy5:635nm,pmt voltage 480V,laser power 30%
Description
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.
