|
| Status |
Public on Jul 17, 2013 |
| Title |
12h-Low_N-2 |
| Sample type |
RNA |
| |
|
| Source name |
12h-Low_Nitrogen-Rep-2
|
| Organism |
Populus tremula x Populus alba |
| Characteristics |
time point: 12h
treatment: low nitrogen
tissue: Roots
genotype: wild type
|
| Treatment protocol |
For low nitrogen treatments the three top internodes of in vitro propagated plants (leaves removed) were placed on filter paper bridges in glass tubes filled with 15 ml liquid ½ MS media for 4 weeks to allow development of root system. Only uniformly developed plants were used in the subsequent experiments. For control (normal nitrogen) treatment we used freshly added ½ MS media containing 20 mM KNO3. For low nitrogen treatment we used ½ MS formulation with 0.05 mM KNO3 (only source of N) and accordingly adjusted the K+ concentration by addition of K2SO4. To maintain constant concentration of the nitrogen in the solution, the media was changed after 2, 5 and 15 days. Roots were sampled at 6, 12, 24, 48, and 408h after transfer to control and low nitrogen media and stored at -80oC until further processed. Total RNA from the roots was extracted as previously described (Busov et al., 2003) and on-column DNaseI-treated (Qiagen).
|
| Growth protocol |
In vitro conditions
|
| Extracted molecule |
total RNA |
| Extraction protocol |
We isolated RNA from about 0.3g tissue. RNA was extracted as previously described (Busov VB,at al, 2003, Plant Physiol 132: 1283-1291) using QIAGEN kits.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard one_cycle Affymetrix protocol from 2 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
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| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Poplar Genome Array. GeneChips were washed and stained in the Affymetrix GeneChip® Fluidics Station 450
|
| Scan protocol |
Arrays were scanned with an Affymetrix GeneChip® Scanner 3000 at 570nm. The Affymetrix eukaryotic hybridization control kit and Poly-A RNA control kit were used to ensure efficiency of hybridization and cRNA amplification. All cRNA was synthesized at the same time. Hybridizations were conducted with one replicate of all times and treatments concurrently. Each array image was visually screened to discount for signal artifacts, scratches or debris.
|
| Description |
Gene expression data from roots
Populus tremula x Populus alba INRA 717-IB4
|
| Data processing |
Microarray data were normalized with Robust Multichip Average (RMA). Rank Product (RP) (Breitling, Armengaud et al. 2004) was used for identifying differentially expressed genes (DEGs) and Benjamini and Hochberg False Discovery Rate (Benjamini and Hochberg 1995) was used to defined those genes that have the corrected p-values (< 0.05) as differentially expressed genes.
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| |
|
| Submission date |
Dec 27, 2012 |
| Last update date |
Jul 17, 2013 |
| Contact name |
Victor Busov |
| E-mail(s) |
[email protected]
|
| Organization name |
Michigan Technological University
|
| Department |
Forest Resources and Environmental Science
|
| Street address |
1400 Townsend Drive
|
| City |
Houghton |
| State/province |
MI |
| ZIP/Postal code |
49931-1295 |
| Country |
USA |
| |
|
| Platform ID |
GPL4359 |
| Series (1) |
| GSE43162 |
Expression data from poplar roots under nitrogen limitation |
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