Pool of extract, NT-1:1.5ug, NT-2:1.5ug, NT-3:1.5ug. Total RNA Isolation - Guanidinium thiocyanate method Reagents All the solutions should be treated with DEPc 2 ml/L Guanidinium solution 4M stock (3 months on bench) 250.0 g Guanidinium (Fluka) 293.0 ml H2O 17.6 ml 0.75 M sodium citrate pH7 26.4 ml 10% sarcosyl 200 ml total Denaturing Guanidinium solution 0.36 ml 2-mercaptoethanol for 50 ml of the stock solution CsCl buffer For 100 ml Weigh 95.97 g CsCl, dissolved it in 0.1 M EDTA to 100 ml, add 200 µl DEPc Weigh the bottle and autoclave 20', check the weigh Protocol Grind 1g of frozen tissue in a mortar under liquid nitrogen to a fine powder. Transfer the powder with a cold spatula in a homogenizer containing 7 ml of the denaturing solution. Mix well by vortexing. Put the mixture to a centrifuge tube to spin down the membranes and debris 10 000 rpm for 10' Load the supernatant onto a 5.7M CsCl cushion (3ml) equilibrated with EDTA. Centrifuge for at least 15 h at 35 000 rpm at 19°C. Remove most of the solution, invert quickly the tube and let it dry for 10'. Cut off the bottom of the tube and dissolve the RNA pellet with100 µl H2O. Add the same volume of phenol-chloroforme, mix well and centrifuge for 3'. Collect the aqueous phase and re-extract the organic phase with 100 µl H2O. Collect the two fractions and re-extract twice with chloroform. Precipitate the RNA with 0.3 M NaAcetate final concentration and 2.5 volume of ethanol for at least 1 h. Spin down the precipitate at 13 000 rpm, 4 °C, 20' and wash the pellet twice with 70% ethanol. Dry the pellet and dissolve it in 100 µl H2O in order to measure the concentration. Keep the RNA at -70°C.
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:Cold - abiotic stress - temperature,temperature:quantity 4degree time 3hour . 12-day-old seedlings were removed from 0.5 x MS agar plates and transferred onto 0.5xMS liquid medium. After 3 h treatment at 4degre Celsius, seedlings were collected and frozen immediately.
Pool of extract, Cold-1:1.5ug, Cold-2:1.5ug, Cold-3:1.5ug. Total RNA Isolation - Guanidinium thiocyanate method Reagents All the solutions should be treated with DEPc 2 ml/L Guanidinium solution 4M stock (3 months on bench) 250.0 g Guanidinium (Fluka) 293.0 ml H2O 17.6 ml 0.75 M sodium citrate pH7 26.4 ml 10% sarcosyl 200 ml total Denaturing Guanidinium solution 0.36 ml 2-mercaptoethanol for 50 ml of the stock solution CsCl buffer For 100 ml Weigh 95.97 g CsCl, dissolved it in 0.1 M EDTA to 100 ml, add 200 µl DEPc Weigh the bottle and autoclave 20', check the weigh Protocol Grind 1g of frozen tissue in a mortar under liquid nitrogen to a fine powder. Transfer the powder with a cold spatula in a homogenizer containing 7 ml of the denaturing solution. Mix well by vortexing. Put the mixture to a centrifuge tube to spin down the membranes and debris 10 000 rpm for 10' Load the supernatant onto a 5.7M CsCl cushion (3ml) equilibrated with EDTA. Centrifuge for at least 15 h at 35 000 rpm at 19°C. Remove most of the solution, invert quickly the tube and let it dry for 10'. Cut off the bottom of the tube and dissolve the RNA pellet with100 µl H2O. Add the same volume of phenol-chloroforme, mix well and centrifuge for 3'. Collect the aqueous phase and re-extract the organic phase with 100 µl H2O. Collect the two fractions and re-extract twice with chloroform. Precipitate the RNA with 0.3 M NaAcetate final concentration and 2.5 volume of ethanol for at least 1 h. Spin down the precipitate at 13 000 rpm, 4 °C, 20' and wash the pellet twice with 70% ethanol. Dry the pellet and dissolve it in 100 µl H2O in order to measure the concentration. Keep the RNA at -70°C.
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
NT Cy5 / Cold 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
Role of PLDzeta to abiotic stress adaptation
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.
