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Status |
Public on Nov 30, 2013 |
Title |
barley_genotype:Kold_day0_pooled |
Sample type |
RNA |
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Source name |
primary shoot
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Organism |
Hordeum vulgare |
Characteristics |
genotype: Kold frost tolerance grade: tolerant developmental stage: LP.03 three leaves visible
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Treatment protocol |
Cold treatment at 2°C was continued for 21 days without any change in the other growth conditions.
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Growth protocol |
After germination at room temperature for 3 days, the seedlings were cultivated for 10 days in hydroponic cultivation systems filled with half-strength modified Hoagland nutrient solution (in order to avoid the limited water availability to plants grown in soil at low temperature; Kocsy et al. 2000) placed in a growth chamber (Controlled Env. Ltd, Winnipeg, Canada) at 18/15°C day/night temperature and 75% relative humidity, with 16 h illumination at 270 µmol m-2 s-1 (Tischner et al. 1997).
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated from 200 µg by the TRIzol reagent (Invitrogen) and mRNA was isolated from 36 µg total RNA with the Dynabeads mRNA purification kit (Dynal).
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Label |
33P
|
Label protocol |
RNA was reserve-transcripted with SuperScript-II rerverse transcriptase kit (Invitrogen) according manufacture's instructions. The cDNA was labeled with MegaPrime random priming kit (Amersham GE Healthcare) and 10 Ci/mL 33-P dCTP (Hartmann-Analytik, Germany)
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Hybridization protocol |
The filters were prehybridized for 4 h at 65°C with 100 µl salmon sperm (10 mg/ml, Stratagene) in 10 mL hybridization buffer (0.5 M Na-phosphate buffer pH 7.0, 2 mM EDTA pH 8.0, 1% BSA, 7% SDS) and then hybridized with denatured radioactive probes for 16 h at 65°C. Homologous barley probes were washed two times with 0.1% SDS + 0,1x SCC for 20 min. Heterologous rye and wheat probes were washed less stringent both for 20 min with solutions containing 0.1% SDS + 1x SSC and 0.1% SDS + 0.5x SCC.
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Scan protocol |
The filters were exposed for 48 h to an imaging screen (Fuji Film) ans signal were detected by the phosphor imaging system FLA-3000 (Fuji Film) at 16 bits/pixel with pixel size of 50 µm. Scanned images were processed with the ArrayVision software from GE Healthcare to quantify signal intensities.
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Description |
b_00_15
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Data processing |
Background-substracted signals were normalized by the quantil normalization method proposed by Bolstad et al. (2003) and mean values were calculated from each double spot.
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Submission date |
Jun 12, 2013 |
Last update date |
Nov 30, 2013 |
Contact name |
Athmer Benedikt |
E-mail(s) |
benedikt.athmer@gmail.com
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Organization name |
IPK Gatersleben
|
Street address |
Correnstrasse 3
|
City |
Gatersleben |
ZIP/Postal code |
06466 |
Country |
Germany |
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Platform ID |
GPL17286 |
Series (1) |
GSE47882 |
Differential gene expression during cold-acclimation in barley, wheat and rye |
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