|
| Status |
Public on Nov 30, 2013 |
| Title |
rye_genotype:LP2_day1_pooled |
| Sample type |
RNA |
| |
|
| Source name |
primary shoot
|
| Organism |
Secale cereale |
| Characteristics |
genotype: LP2
frost tolerance grade: tolerant
developmental stage: LP.03 three leaves visible
|
| Treatment protocol |
Cold treatment at 2°C was continued for 21 days without any change in the other growth conditions.
|
| 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).
|
| 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).
|
| 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)
|
| |
|
| 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.
|
| 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.
|
| Description |
r_01_02
|
| 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.
|
| |
|
| Submission date |
Jun 12, 2013 |
| Last update date |
Nov 30, 2013 |
| Contact name |
Athmer Benedikt |
| E-mail(s) |
benedikt.athmer@gmail.com
|
| Organization name |
IPK Gatersleben
|
| Street address |
Correnstrasse 3
|
| City |
Gatersleben |
| ZIP/Postal code |
06466 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17286 |
| Series (1) |
| GSE47882 |
Differential gene expression during cold-acclimation in barley, wheat and rye |
|
