|
| Status |
Public on Oct 01, 2016 |
| Title |
561827A04 - Tilted_upper_BR2 vs Control_upper_BR2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control_upper_BR2
|
| Organism |
Populus tremula x Populus alba |
| Characteristics |
treatment: control
face of the stem: upper
cultivar: 717-1b4
age: 73 days
|
| Treatment protocol |
Name:Light - abiotic stress - light,light:time 30min . young poplar trees were staked one week before use. For the experiment, the plants were placed in an isotropic light environment and were tilted at 35° during 30 min. Control plants were kept straight in the isotropic light device. Xylem samples were collected from the upper and lower face of the stems.
|
| Growth protocol |
stem - Media=potting soil hygrometry=N/A Temperature=24°C (Day) / 18°C (night) Light=16h (Day) / 8h (night)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Control_upper_BR2:15ug. (Qiagen_RNeasy_Micro_Handbook.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 |
Tilted_upper_BR2
|
| Organism |
Populus tremula x Populus alba |
| Characteristics |
treatment: tilted
face of the stem: upper
cultivar: 717-1b4
age: 73 days
|
| Treatment protocol |
Name:Light - abiotic stress - light,light:time 30min . young poplar trees were staked one week before use. For the experiment, the plants were placed in an isotropic light environment and were tilted at 35° during 30 min. Control plants were kept straight in the isotropic light device. Xylem samples were collected from the upper and lower face of the stems.
|
| Growth protocol |
stem - Media=potting soil hygrometry=N/A Temperature=24°C (Day) / 18°C (night) Light=16h (Day) / 8h (night)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tilted_upper_BR2:15ug. (Qiagen_RNeasy_Micro_Handbook.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 |
Control_upper_BR2 Cy5 / Tilted_upper_BR2 Cy3 : 30pmol.
|
| Scan protocol |
Mapix, Cy3:pmt voltage 532nm,480V,laser power 35%, Cy5:635nm,pmt voltage 480V,laser power 30%
|
| Description |
The experiments were designed to identify genes responding to the gravitropic stimulus in the xylem of poplar stem independently of phototropic responses.
|
| 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 |
Sep 15, 2016 |
| Last update date |
Oct 01, 2016 |
| 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 |
| |
|
| Platform ID |
GPL17461 |
| Series (1) |
| GSE86951 |
gravitropic stimulation / isotropic light-Characterization of early molecular events of woody stem gravitropism independently of phototropic responses |
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