leaf - Media : 2(KNO3)/Ca(NO3)2 = 1.5mM MgSO4/KH2PO4 = 2 mM K2SO4 = 1 mM CaCl2 = 0.7 mM Oligo(Cooc and Lesaint , 1975)= 1 ml.L-1 Fer-EDTA = 10 mg.L-1 hygrometry : 70 % Temperature : day : 21degreeC, night :17 degreeC Light : 8 h WS Arabidopsis ecotype and colza were grown on 6mM nitrate as sole nitrogen source during 35 days under short days . At T0, plants were then starved for nitrate for 10 days and root and shoot samples were harvested separately 2 and 10 days after treatment (T2, T10). Then, nitrate (6 mM) was re-supplied for 1 and 24 hours (T+1, T+24).
Extracted molecule
total RNA
Extraction protocol
F_C15j Rapeseed:5ug.
Label
Cy5
Label protocol
labelling Cy3 and Cy5 indirect, amplification=yes, DNA 5 ug. 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).
leaf - Media : 2(KNO3)/Ca(NO3)2 = 1.5mM MgSO4/KH2PO4 = 2 mM K2SO4 = 1 mM CaCl2 = 0.7 mM Oligo(Cooc and Lesaint , 1975)= 1 ml.L-1 Fer-EDTA = 10 mg.L-1 hygrometry : 70 % Temperature : day : 21degreeC, night :17 degreeC Light : 8 h WS Arabidopsis ecotype and colza were grown on 6mM nitrate as sole nitrogen source during 35 days under short days . At T0, plants were then starved for nitrate for 10 days and root and shoot samples were harvested separately 2 and 10 days after treatment (T2, T10). Then, nitrate (6 mM) was re-supplied for 1 and 24 hours (T+1, T+24).
Extracted molecule
total RNA
Extraction protocol
F_T0 Rapeseed:5ug.
Label
Cy3
Label protocol
labelling Cy3 and Cy5 indirect, amplification=yes, DNA 5 ug. 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
F_C15j Rapeseed Cy5 / F_T0 Rapeseed Cy3 : 30pmol. 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
GenePix Pro 3.0, Cy3:pmt voltage 532nm,650V,laser power 100%, Cy5:635nm,pmt voltage 620V,laser power 100%
Description
What are the transcriptomic short- and long-term plant responses to nitrogen starvation and nitrogen re-supply?
Data processing
The raw data comprised the logarithm of median feature pixel intensity at wavelength 635 nm (red) and 532 nm (green). No background was subtracted. An array-by-array normalization was performed to remove systematic biases. First, we excluded spots that were considered badly formed features by the experimenter (Flags=-100). Then we performed a global intensity-dependent normalization using the loess procedure (see Yang et al., 2002) to correct the dye bias. Finally, on each block, the log-ratio median is subtracted from each value of the log-ratio of the block to correct a print-tip effect on each metablock.
