|
| Status |
Public on Jul 01, 2008 |
| Title |
13415337 - penta vs WT |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
WT
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
Arabidopsis thaliana (landsberg erecta) - dev.stage (Boyes et al. Plant Cell 2001):boyes 1.02
|
| Treatment protocol |
Name:control - genetic treatment - o nacl,nacl 200 mm:time 0 . NaCl was added to MS liquid (final concentration 200mM) for 30 min or 1 h. For control, no salt was added. Treatment was stopped by freezing in liquid nitrogen.
|
| Growth protocol |
plantlet - Media: MS agar for 9 days then MS liquid for 3 days then MS liquid + NaCL 200mM or control hygrometry 55% Temperature 22degreeC Light 16h/day
|
| Extracted molecule |
total RNA |
| Extraction protocol |
WT:30ug.
|
| 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).
|
| |
|
| Channel 2 |
| Source name |
penta
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
Arabidopsis thaliana (landsberg erecta) mutant (ga1-3 gai-t6 rga-t2rgl1-1 rgl2-1) - dev.stage (Boyes et al. Plant Cell 2001):boyes 1.02
|
| Treatment protocol |
Name:control - genetic treatment - o nacl,nacl 200 mm:time 0 . NaCl was added to MS liquid (final concentration 200mM) for 30 min or 1 h. For control, no salt was added. Treatment was stopped by freezing in liquid nitrogen.
|
| Growth protocol |
plantlet - Media: MS agar for 9 days then MS liquid for 3 days then MS liquid + NaCL 200mM or control hygrometry 55% Temperature 22degreeC Light 16h/day
|
| Extracted molecule |
total RNA |
| Extraction protocol |
penta:30ug.
|
| 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 |
WT Cy5 / penta Cy3 : 30pmol.
|
| Scan protocol |
GenePix Pro 6.0, Cy3:pmt voltage 532nm,500V,laser power 30%, Cy5:635nm,pmt voltage 500V,laser power 30%
|
| Description |
Identification of DELLA-dependent dowtream targets in response to salt stress
|
| 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.
|
| |
|
| Submission date |
Jul 23, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Véronique BRUNAUD |
| E-mail(s) |
veronique.brunaud@inrae.fr
|
| Organization name |
INRA - CNRS - UPSUD
|
| Lab |
IPS2
|
| Street address |
rue Noetzlin
|
| City |
Gif-sur-Yvette |
| ZIP/Postal code |
91190 |
| Country |
France |
| |
|
| Platform ID |
GPL5337 |
| Series (1) |
| GSE8556 |
della-regulation of salt stress responses-DELLAs contribute to salt stress responses |
|
