whole plant - Plants were germinated and grown in soil under a long day (16h light / 8h dark) light regime. Hygrometry of the greenhouse was 70 % and temperature was 22degreeC.
Extracted molecule
total RNA
Extraction protocol
ntr-3:12ug. Extraction protocol: Plant tissue was ground to fine powder using liquid nitrogen in a 2mL eppendorf tube. The samples were homogenized in TRIzol Reagent (Invitrogen) 1 mL for 50-100 mg of plant tissue, by vortexing and incubated at room temperature for 5 min. Chloroform (200µl/1mL TRIzol) was added and the capped tubes were hand-shaken for 15 sec. They were incubated for 2-3 min, then centrifuged for 15 min at 11000 rpm at 4°C. The aqueous phase was transferred to a new 1.5 mL tube and 500ul of isopropanol was added. The samples were incubated for 10 min at room temperature then centrifuged for 10 min at 11000 rpm at 4°C. The supernatant was removed and the pellet was washed with 1 mL of 75% ethanol in DEPC-treated water. The samples were centrifuged for 5 min at 11000 rpm at 4°C. The pellet was air-dried for 10 min at room-temperature, then resuspended in 500 µl DEPC-treated water, and precipated with 2 volumes of 100 % ethanol and 1/10 V of Sodium acetate 3M, pH5,2. The samples were precipitated 30 min at -20 °C and centrifuged for 15 min at 10000 rpm at 4°C. The supernatant was removed, the pellet was air-dried and resuspended in DEPC-treated water. The RNA solution was purified on Qiagen RN-Easy column. The RNA quality was then acessed on Agilent Bioanalyser following the manufacturer's protocol and the quantity is determined by Ribogreen
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).
whole plant - Plants were germinated and grown in soil under a long day (16h light / 8h dark) light regime. Hygrometry of the greenhouse was 70 % and temperature was 22degreeC.
Extracted molecule
total RNA
Extraction protocol
col-3:9.6ug. Extraction protocol: Plant tissue was ground to fine powder using liquid nitrogen in a 2mL eppendorf tube. The samples were homogenized in TRIzol Reagent (Invitrogen) 1 mL for 50-100 mg of plant tissue, by vortexing and incubated at room temperature for 5 min. Chloroform (200µl/1mL TRIzol) was added and the capped tubes were hand-shaken for 15 sec. They were incubated for 2-3 min, then centrifuged for 15 min at 11000 rpm at 4°C. The aqueous phase was transferred to a new 1.5 mL tube and 500ul of isopropanol was added. The samples were incubated for 10 min at room temperature then centrifuged for 10 min at 11000 rpm at 4°C. The supernatant was removed and the pellet was washed with 1 mL of 75% ethanol in DEPC-treated water. The samples were centrifuged for 5 min at 11000 rpm at 4°C. The pellet was air-dried for 10 min at room-temperature, then resuspended in 500 µl DEPC-treated water, and precipated with 2 volumes of 100 % ethanol and 1/10 V of Sodium acetate 3M, pH5,2. The samples were precipitated 30 min at -20 °C and centrifuged for 15 min at 10000 rpm at 4°C. The supernatant was removed, the pellet was air-dried and resuspended in DEPC-treated water. The RNA solution was purified on Qiagen RN-Easy column. The RNA quality was then acessed on Agilent Bioanalyser following the manufacturer's protocol and the quantity is determined by Ribogreen
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
ntr3 Cy5 / Col3 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,700V,laser power 100%, Cy5:635nm,pmt voltage 650V,laser power 100%
Description
Thioredoxins are small redox proteins implicated in crucial pathways of cell life. They are reduced by a conserved flavoprotein named NADPH-dependent thioredoxin reductase (NTR). In order to give an insight into the functions of the NTR/thioredoxin pathway in Arabidopsis, we have used a reverse genetic approch. Both NTR genes (NTRA and NTRB) have been inactivated in the double ntra ntrb mutant. T-DNA lines have been provided by the SALK library. Surprisingly, this mutant shows a limited phenotype suggested that some additional redox pathways are involved in compensating the inactivation of the NTR/thioredoxin pathway. In order to isolate compensatory genes that would be transcriptionally induced in the ntra ntrb mutant, we have performed this CATMA project.
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.
