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).
Name:Nitrate_24h - environmental treatment - azote concentration,nitrate:quantity 6mM time 24hour . 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).
Growth protocol
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+24h 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).
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.
