Name:C-Urea - compound based treatment - compound addition,urea:quantity 1mM time 7day . 7 days on : Media : Urea 1 mM MgSO4/KH2PO4 1 mM K2SO4 2.5 mM CaCl2 2.2 mM Fer Sequestrene 10 mg.L-1 Oligo 1 ml.L-1 hygrometry : 80% Temperature : Day 21degre Celsius/Night 17degre Celsius Light : 8 h/1h
Growth protocol
root - NH4NO3 0.5 mM MgSO4/KH2PO4 1 mM K2SO4 2.5 mM CaCl2 2.2 mM Fer Sequestrene 10 mg.L-1 Oligo 1 ml.L-1 hygro 80% Temp : Day 21degreeC/Night 17degreeC, light : Day 8 h/night 16h
Extracted molecule
total RNA
Extraction protocol
RC-H2:17ug. Grind samples in liquide nitrogen, Heat 500 µl phenol pH4+500 µl TLES at 80°C, homogenize samples in hot phenol+TLES, vortex 30 '', heat again at 80°C 1', add 500 µl chloroforme/isoamylalcool (24:1), vortex 30'', transfer in safelock 2 ml and put on ice, centrifuge 10' 15000 rpm 4°C, transfer supernatant in eppendorf and add the same volume of LiCl 4M (about 400 µl), precipitate on ice overnight at 4°C. Centrifuge 30' 14500 rpm 4°C, remove supernatant, resuspend the pellet in 250 µl RNase-free water, add 1/10 volume of sodium acetate 2.5M pH 5.6 and 2 volumes of 100% ethanol, precipitate overnight at - 20°C. Centrifuge 20' 15000 rpm 4°C, wash the RNA pellet with 500 µl of 70% ethanol, centrifuge 10' 15000 rpm 4°C, briefly dry the pellet (air dry for 5-10'), resuspend in 30 µl RNase-free water.
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:A-Ammonitrate - compound based treatment - compound addition,ammonitrate:quantity 1mM time 7day . 7 days on : Media : NH4NO3 1 mM MgSO4/KH2PO4 1 mM K2SO4 2.5 mM CaCl2 2.2 mM Fer Sequestrene 10 mg.L-1 Oligo 1 ml.L-1 hygrometry : 80% Temperature : Day 21degre Celsius/Night 17degre Celsius Light : 8 h/1h
Growth protocol
root - NH4NO3 0.5 mM MgSO4/KH2PO4 1 mM K2SO4 2.5 mM CaCl2 2.2 mM Fer Sequestrene 10 mg.L-1 Oligo 1 ml.L-1 hygro 80% Temp : Day 21degreeC/Night 17degreeC, light : Day 8 h/night 16h
Extracted molecule
total RNA
Extraction protocol
RA-H2:20ug. Grind samples in liquide nitrogen, Heat 500 µl phenol pH4+500 µl TLES at 80°C, homogenize samples in hot phenol+TLES, vortex 30 '', heat again at 80°C 1', add 500 µl chloroforme/isoamylalcool (24:1), vortex 30'', transfer in safelock 2 ml and put on ice, centrifuge 10' 15000 rpm 4°C, transfer supernatant in eppendorf and add the same volume of LiCl 4M (about 400 µl), precipitate on ice overnight at 4°C. Centrifuge 30' 14500 rpm 4°C, remove supernatant, resuspend the pellet in 250 µl RNase-free water, add 1/10 volume of sodium acetate 2.5M pH 5.6 and 2 volumes of 100% ethanol, precipitate overnight at - 20°C. Centrifuge 20' 15000 rpm 4°C, wash the RNA pellet with 500 µl of 70% ethanol, centrifuge 10' 15000 rpm 4°C, briefly dry the pellet (air dry for 5-10'), resuspend in 30 µl RNase-free water.
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
RC-H2 Cy5 / RA-H2 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 plant responses to urea nitrogen 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.
