|
| Status |
Public on Jun 01, 2015 |
| Title |
WT stems, MS6 media - Biological Replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
wild-type stem tissues, grown on MS6 PGRs-depleted medium
|
| Organism |
Populus tremula x Populus tremuloides |
| Characteristics |
tissue: stem
genotype/variation: WT
media: MS6
|
| Treatment protocol |
Five-week-old, in vitro grown stem segments were collected from wild-type and transgenic line 35S::POPACAULIS5-B2, grown on MS6 medium. Two week-old stem segments were collected from wild-type and 35S::POPACAULIS5-B2 transgenic plants, grown on MS2 medium. Sampled tissues were directly frozen in liquid nitrogen when collected all at the same time in the same day and stored at -80ºC. Three pools of stem segments from three individual plants (nine in total) from each line (wild-type and transgenic) in both growth conditions (MS6 and MS2) were used, constituting three biological replicates for each pair genotype/growth condition.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNeasy Plant Mini Extraction kit (Qiagen) following the manufacturer´s instructions
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA according to manufacturer´s instructions. RNA was processed for use on Affymetrix GeneChip Poplar Genome Arrays, according to the manufacturer’s GeneChip 3′ IVT Express kit user manual. 100 ng of total RNA containing spiked in Poly-A RNA controls was used in a reverse transcription reaction (GeneChip 3′ IVT Express Kit; Affymetrix) to generate first-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in a 16 h in vitro transcription (IVT) reaction to generate aRNA (GeneChip 3′ IVT Express Kit; Affymetrix). Size distribution of the aRNA and fragmented aRNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay.
|
| |
|
| Hybridization protocol |
15 μg of fragmented aRNA was used containing added hybridization controls. The mixture was hybridized on arrays for 16 h at 45°C. Standard post hybridization wash and double-stain protocols (FS450_0004; GeneChip HWS kit, Affymetrix) were used on an Affymetrix GeneChip Fluidics Station 450.
|
| Scan protocol |
Arrays were scanned on an Affymetrix GeneChip scanner 3000 7 G.
|
| Description |
Gene expression data from stem tissues of wild-type hybrid aspen grown on MS6 PGRs-depleted medium
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings to obtain Absent/Present calls and to assure that all quality parameters were in the recommended range. Subsequent analysis was carried out with DNA-Chip Analyzer (dChip) 2010 (http://www.dchip.org, Cheng Li Lab, Harvard). Arrays were normalized to a baseline array with median CEL intensity by applying an Invariant Set Normalization Method.
|
| |
|
| Submission date |
Apr 08, 2013 |
| Last update date |
Jun 01, 2015 |
| Contact name |
Ana Milhinhos |
| E-mail(s) |
milhinho@itqb.unl.pt
|
| Phone |
00351214469627
|
| Organization name |
IBET ITQB-UNL
|
| Department |
Plant Sciences
|
| Lab |
Forest Biotech Lab
|
| Street address |
Av Republica EAN Oeiras
|
| City |
Oeiras |
| State/province |
Select a State or Province |
| ZIP/Postal code |
2780 |
| Country |
Portugal |
| |
|
| Platform ID |
GPL4359 |
| Series (1) |
| GSE45879 |
Thermospermine-induced transcriptomic changes in Populus stem |
|
