Name:Cadmium - compound based treatment - compound addition,cadmium:quantity .2mM time 12hour . After 5 days of growth in a fresh medium (5% PCV at day0), CdCl2 was added to tested cells (Cad) to a final concentration of 200(u)M. Nothing was added to control cells (Tem). After 12 hours of growth +/- cadmium, cells were harvested and frozen in liquid nitrogen.
Growth protocol
cell culture - Murashige and Skoog salts (Sigma), 0.5 mM kinetin, 0.34 mM 2-4D, vitamins mix (4 mM Nicotinic Acid, 1.26 uM Calcium Dpantothenate, 2.66 mM Glycine, 150 uM Thiamine-HCl, 110 uM Folic acid, 0.25 mM Pyridoxine-HCl, 20 uM Biotine and 28 mM Myo-inositol) and 3 % sucrose with pH adjusted to 5.6
Extracted molecule
total RNA
Extraction protocol
Polys Cd 2:5ug. Total RNA were extracted with 2 mL/g-1 extraction buffer (0,1M NaCl, 2% SDS, 50mM TrisHCl pH 9, 10mM EDTA, 20mM b-mercapto-ethanol) and 5mL acid phenol and 5mL chloroform/isoamylic alcohol (24/1, v/v). After centrifuge 10 min at 8000g, aqueous phases was extracted one more time in 5mL acid phenol and 5mL chloroform/isoamylic alcohol. After last centrifuge, aqueous phase in a fresh tube was added to Na acetate 3M pH3,1 and 3 vol of ethanol 100% ON -20°C. RNAs were precipitated by centrifugation (30min 16000g). Pellet was resuspended in water. After quantification by spectrophotometry, 150µg of total RNA were purified on Promega Wizard columns
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:Cadmium - compound based treatment - compound addition,cadmium:quantity .2mM time 12hour . After 5 days of growth in a fresh medium (5% PCV at day0), CdCl2 was added to tested cells (Cad) to a final concentration of 200(u)M. Nothing was added to control cells (Tem). After 12 hours of growth +/- cadmium, cells were harvested and frozen in liquid nitrogen.
Growth protocol
cell culture - Murashige and Skoog salts (Sigma), 0.5 mM kinetin, 0.34 mM 2-4D, vitamins mix (4 mM Nicotinic Acid, 1.26 uM Calcium Dpantothenate, 2.66 mM Glycine, 150 uM Thiamine-HCl, 110 uM Folic acid, 0.25 mM Pyridoxine-HCl, 20 uM Biotine and 28 mM Myo-inositol) and 3 % sucrose with pH adjusted to 5.6
Extracted molecule
total RNA
Extraction protocol
Monos Cad 2:5ug. Total RNA were extracted with 2 mL/g-1 extraction buffer (0,1M NaCl, 2% SDS, 50mM TrisHCl pH 9, 10mM EDTA, 20mM b-mercapto-ethanol) and 5mL acid phenol and 5mL chloroform/isoamylic alcohol (24/1, v/v). After centrifuge 10 min at 8000g, aqueous phases was extracted one more time in 5mL acid phenol and 5mL chloroform/isoamylic alcohol. After last centrifuge, aqueous phase in a fresh tube was added to Na acetate 3M pH3,1 and 3 vol of ethanol 100% ON -20°C. RNAs were precipitated by centrifugation (30min 16000g). Pellet was resuspended in water. After quantification by spectrophotometry, 150µg of total RNA were purified on Promega Wizard columns
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
Polys Cd 2 Cy5 / Monos Cd 2 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
Is there a change in the translation in Cadmium stress condition ?
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.
