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Sample GSM212603 Query DataSets for GSM212603
Status Public on Jul 01, 2008
Title 13415202 - ga1-3_2 vs WT2
Sample type RNA
 
Channel 1
Source name WT2
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 WT2:30ug.
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 ga1-3_2
Organism Arabidopsis thaliana
Characteristics Arabidopsis thaliana (landsberg erecta) mutant (ga1-3) - 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 ga1-3_2:30ug.
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 WT2 Cy5 / ga1-3_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 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 INRAE - 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

Data table header descriptions
ID_REF ID number
VALUE Normalized log2 ratio median intensity of Ch2(Cy3)/Ch1(Cy5) (Ch1=reference)

Data table
ID_REF VALUE
1 0.3269
2 0.4948
259 0.1071
260 -0.0322
261 -0.149
262 0.2339
263 0.2025
265 -0.1052
266 0.2141
267 0.2229
268 -0.2165
269 -0.1611
270 -0.2273
271 -0.2308
272 -0.3177
273 0.1458
274 -0.3118
275 -0.0528
276 -0.0005
277 0.2809

Total number of rows: 24366

Table truncated, full table size 308 Kbytes.




Supplementary file Size Download File type/resource
GSM212603.gpr.gz 1.8 Mb (ftp)(http) GPR
Processed data included within Sample table

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