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| Status |
Public on Mar 28, 2012 |
| Title |
13688508 - ctrl 4 degree Celsius 30min vs DMS 4 degree Celsius 30min |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
DMS 4 degree Celsius 30min
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: columbia
harvest date: 11-09-07
treatment: DMS 4 degree Celsius 30min
|
| Treatment protocol |
Name:DMS 4degre Celsius 30min - compound based treatment - temperature,dms:time 30min . DMS was added 30 min before cold choc. A t T0, cells were transfered at 00c for 30 min and directly harvested and N2 frozen before RNA extraction.
|
| Growth protocol |
cell culture/primary cell - Media=2-4D liquid medium hygrometry=non relevant Temperature=22°c Light=40 mmole photon s-1 m-2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Pool of extract, DMS 4degre Celsius 30min set1:0.33ug, DMS 4degre Celsius 30min set2:0.33ug, DMS 4degre Celsius 30min set3:0.33ug. (RS07-05_Sphingolipids-cold_Extraction-protocol.doc)
|
| 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).
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| |
|
| Channel 2 |
| Source name |
ctrl 4 degree Celsius 30min
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: columbia
harvest date: 25-09-07
treatment: DMSO 4 degree Celsius 30min
|
| Treatment protocol |
Name:DMSO 4degre Celsius 30min - compound based treatment - temperature,dmso:time 30min . DMSO was added 30 min before cold choc. A t T0, cells were transfered at 00c for 30 min and directly harvested and N2 frozen before RNA extraction.
|
| Growth protocol |
cell culture/primary cell - Media=2-4D liquid medium hygrometry=non relevant Temperature=22°c Light=40 mmole photon s-1 m-2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Pool of extract, ctrl 4degre Celsius 30min set1:0.33ug, ctrl 4degre Celsius 30min set2:0.33ug, ctrl 4degre Celsius 30min set3:0.33ug. (RS07-05_Sphingolipids-cold_Extraction-protocol.doc)
|
| 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).
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| |
|
| |
| Hybridization protocol |
DMS 4degre Celsius 30min Cy5 / ctrl 4degre Celsius 30min 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 |
GenePix Pro 6.0, Cy3:pmt voltage 532nm,500V,laser power 30%, Cy5:635nm,pmt voltage 500V,laser power 30%
|
| Description |
The cold choc response seems to be partly triggered by Sphingolipid species. To date no gene response as been associated to sphingolipid signaling pathway in plant. Our aim is to identify among the cold induced genes the ones regulated by sphingolipids and to try to define a sphingolipid pathway specific group of genes.
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| 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.
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| Submission date |
Mar 21, 2012 |
| Last update date |
Mar 28, 2012 |
| Contact name |
Soubigou-Taconnat Ludivine |
| E-mail(s) |
soubigou@evry.inra.fr
|
| Organization name |
INRA
|
| Department |
URGV
|
| Lab |
ADT
|
| Street address |
2 rue gaston crémieux
|
| City |
evry |
| ZIP/Postal code |
91000 |
| Country |
France |
| |
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| Platform ID |
GPL4346 |
| Series (1) |
| GSE36659 |
sphingo-1-Identification of Sphingolipid regulated genes in cold stress response. Sphingolipids regulated genes. |
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