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Sample GSM770193 Query DataSets for GSM770193
Status Public on Dec 31, 2011
Title 14248996 - WT + Fe 1 vs WT - Fe 1
Sample type RNA
 
Channel 1
Source name WT - Fe 1
Organism Arabidopsis thaliana
Characteristics ecotype: columbia
genotype/variation: wildtype
age: 6day
dev.stage (boyes et al. plant cell 2001): boyes: 1.00
treatment: no Fe
Treatment protocol Name:without Fe - environmental treatment - fe deficiency,fe:quantity 0uM time 6day . Seedlings were germinated and grown in the presence of 50 µM Fe or absence of Fe (0 µM) on Hoagland medium agar plates until the age of 6 days. Whole seedlings were harvested for transcriptome analysis, in a total of three biological replicates.
Growth protocol whole plant - Media : Hoagland hygrometry : sterile plate Temperature : 21°C Light : 16h light
Extracted molecule total RNA
Extraction protocol WT - Fe 1:5ug. (Qiagen_RNeasy.pdf)
Label Cy5
Label protocol labelling Cy3 and Cy5 direct, amplification=yes, aRNA 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 WT + Fe 1
Organism Arabidopsis thaliana
Characteristics ecotype: columbia
genotype/variation: wildtype
age: 6day
dev.stage (boyes et al. plant cell 2001): boyes: 1.00
treatment: Fe
Treatment protocol Name:with Fe - environmental treatment - fe deficiency,fe:quantity 50uM time 6day . Seedlings were germinated and grown in the presence of 50 µM Fe or absence of Fe (0 µM) on Hoagland medium agar plates until the age of 6 days. Whole seedlings were harvested for transcriptome analysis, in a total of three biological replicates.
Growth protocol whole plant - Media : Hoagland hygrometry : sterile plate Temperature : 21°C Light : 16h light
Extracted molecule total RNA
Extraction protocol WT + Fe 1:5ug. (Qiagen_RNeasy.pdf)
Label Cy3
Label protocol labelling Cy3 and Cy5 direct, amplification=yes, aRNA 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 - Fe 1 Cy5 / WT + Fe 1 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,480V,laser power 35%, Cy5:635nm,pmt voltage 480V,laser power 30%
Description Response of ein3eil1 mutants to Fe deficiency
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 Aug 01, 2011
Last update date Dec 31, 2011
Contact name Eddy Blondet
E-mail(s) blondet@evry.inra.fr
Phone 0160874520
Organization name INRA
Department URGV
Lab GFA
Street address 2 rue Gaston crémieux
City Evry
ZIP/Postal code 91057
Country France
 
Platform ID GPL9553
Series (1)
GSE26510 response of ein3eil1 mutants to fe deficiency-Role of ethylene in Fe deficieny response signalling

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

Data table
ID_REF VALUE
1 -0.0972
2 -0.4443
3 -0.4924
4 0.0776
5 -0.6264
6 -0.6243
7 -0.551
8 -0.51
9 0.0944
10 -0.2728
11 -0.2437
12 -0.373
13 0.0049
14 -0.0064
15 -0.4073
16 -0.3844
17 0.3326
18 -0.0111
19 -0.1681
20 -0.2199

Total number of rows: 34642

Table truncated, full table size 441 Kbytes.




Supplementary file Size Download File type/resource
GSM770193.gpr.gz 2.5 Mb (ftp)(http) GPR
Processed data included within Sample table

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