Name:Phyllobacterium-in vitro - environmental treatment - pgpr,phyllobacterium:time 6day . 2.108 cfu Phyllobacterium per ml medium
Growth protocol
root - Media: 0.5 mM CaSO4, 5 mM KNO3, 0.5 mM MgCl2, 1 mM KH2PO4, 50 uM Na2FeEDTA, 50 uM H3Bo3, 15 uM MnCl2, 1 uM ZnCl2, 1 uM CuCl2, 0.03 uM (NH4)6Mo7O24 and 2.5 mM MES, with pH adjusted to 5.7 with KOH; plus agar 0.8 % (v/w). hygrometry : 70% Temperature : 20 degreeC Light : 180 umol m-2 s-1, 16 h per day
Extracted molecule
total RNA
Extraction protocol
Root Phyllo:5.37ug.
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).
root - Media: 0.5 mM CaSO4, 5 mM KNO3, 0.5 mM MgCl2, 1 mM KH2PO4, 50 uM Na2FeEDTA, 50 uM H3Bo3, 15 uM MnCl2, 1 uM ZnCl2, 1 uM CuCl2, 0.03 uM (NH4)6Mo7O24 and 2.5 mM MES, with pH adjusted to 5.7 with KOH; plus agar 0.8 % (v/w). hygrometry : 70% Temperature : 20 degreeC Light : 180 umol m-2 s-1, 16 h per day
Extracted molecule
total RNA
Extraction protocol
Control Root Phyllo:11.82ug.
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
Root Phyllo Cy5 / Control Root Phyllo 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
Experiment 1 : Which genes are up- or down-regulated in Arabidopsis thaliana cultivated in vitro with increased lateral root development in response to Phyllobacterium STM196 inoculation. Experiment 2 : Which genes are up- or down-regulated during the ISR triggered by a rhizobacteria, in comparison with those affected by a pathogenic interaction. Experiment 3 : which genes are specifically induced or repressed in Arabidopsis thaliana by inoculation of the soil with a PGPR vs a bacteria that has the ability to trigger nodule formation in a Legume.
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.
