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Sample GSM1262249 Query DataSets for GSM1262249
Status Public on Feb 01, 2014
Title 557282A03 - Col0_bio3 vs cyp715A1_bio3
Sample type RNA
 
Channel 1
Source name cyp715A1_bio3
Organism Arabidopsis thaliana
Characteristics tissue: flower buds
genotype: cyp715A1 mutant
ecotype: columbia
developmental stage: boyes: 6.30 (Boyes et al. Plant Cell 2001)
Treatment protocol no treatment
Growth protocol flower bud - Media: soil, hygrometry:65%, temperature: 21°c jour et 18°C nuit, light: 12h/12h - 80 µmol.m-2.s-1.
Extracted molecule total RNA
Extraction protocol cyp715A1_bio3:80mg. (NucleoSpinRNAPlant.pdf)
Label Cy5
Label protocol labelling Cy3 and Cy5 direct, amplification=yes, aRNA 5 ug. (Labelling_protocol.txt) 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 Col0_bio3
Organism Arabidopsis thaliana
Characteristics tissue: flower buds
genotype: Col0
ecotype: columbia
developmental stage: boyes: 6.30 (Boyes et al. Plant Cell 2001)
Treatment protocol no treatment
Growth protocol flower bud - Media: soil, hygrometry:65%, temperature: 21°c jour et 18°C nuit, light: 12h/12h - 80 µmol.m-2.s-1.
Extracted molecule total RNA
Extraction protocol Col0_bio3:80mg. (NucleoSpinRNAPlant.pdf)
Label Cy3
Label protocol labelling Cy3 and Cy5 direct, amplification=yes, aRNA 5 ug. (Labelling_protocol.txt) 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 cyp715A1_bio3 Cy5 / Col0_bio3 Cy3 : 30pmol. (Hybridization_Protocol.txt) 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 Mapix, Cy3:pmt voltage 532nm,480V,laser power 35%, Cy5:635nm,pmt voltage 480V,laser power 30%
Description The microarray analysis is part of a project aimed at characterizing the function of the cytochrome P450 CYP715A1 in Arabidopsis thaliana.
Data processing For each array, the raw data comprised the logarithm of median feature pixel intensity at wavelengths Cy5 (red) and Cy3 (green).For each array, a global intensity-dependent normalization using the loess procedure (Yang et al., 2002) was performed to correct the dye bias.Log-ratios are then averaged over the duplicate probes to get a value per gene.
 
Submission date Nov 12, 2013
Last update date Feb 01, 2014
Contact name Stéphanie Pateyron
E-mail(s) pateyron@evry.inra.fr
Organization name IPS2_Institute of Plant Sciences Paris-Saclay
Lab Transcriptomic Plateforme POPS
Street address Rue de Noetzlin _ Batiment 630
City Orsay
ZIP/Postal code 91405
Country France
 
Platform ID GPL17404
Series (1)
GSE52269 col-0 vs cyp715a1-Genome-wide transcriptional analysis of cyp715A1

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
CATMA1OA00010F1 0.292406071
CATMA1OA00010R1 0.429429965
CATMA1OA00020F1 0.192687381
CATMA1OA00020R1 0.165731131
CATMA1OA00030F1 -0.210216633
CATMA1OA00030R1 -0.123805578
CATMA1OA00035F1 -0.23329204
CATMA1OA00035R1 0.13556133
CATMA1OA00040F1 0.047003032
CATMA1OA00040R1 -0.052312584
CATMA1OA00045F1 0.251629692
CATMA1OA00045R1 0.089531403
CATMA1OA00050F1 -0.169432893
CATMA1OA00050R1 -0.00878277
CATMA1OA00060F1 -0.167453624
CATMA1OA00060R1 0.057585124
CATMA1OA00070F1 0.137720607
CATMA1OA00070R1 -0.285865239
CATMA1OA00080F1 0.036963271
CATMA1OA00080R1 0.250858014

Total number of rows: 73228

Table truncated, full table size 2032 Kbytes.




Supplementary file Size Download File type/resource
GSM1262249_557282A03_532.pair.gz 3.9 Mb (ftp)(http) PAIR
GSM1262249_557282A03_635.pair.gz 3.9 Mb (ftp)(http) PAIR
Processed data included within Sample table

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