|
| Status |
Public on Mar 17, 2017 |
| Title |
Wheat 6R Replica 3 Control |
| Sample type |
RNA |
| |
|
| Source name |
Wheat 6R control
|
| Organism |
Triticum aestivum |
| Characteristics |
line: Triticum aestivum secale cereale addition line 6R
age: 5 days
tissue: root apex
exposed to: control solution
|
| Treatment protocol |
4 day old seedlings were transferred to the same nutrient solution with or without 200 μM AlCl3 for 24h.
|
| Growth protocol |
Seeds were sterilized with 10% (v/v) sodium hypochlorite for 10 min, rinsed with deionized water, and incubated on wet filter paper in the dark for 24h at 4ºC and 24h at room temperature. Seedlings were placed on a styrofoam sheet with a polyethylene net bottom. The styrofoam sheets were floated on a nutrient solution in a plastic tray. Seedlings were grown in aerated medium under controlled environmental conditions (25°C and a 16h photoperiod). Seedlings were grown in the medium described by Aniol, 1981, with the modification of Gallego and Benito (1997). The nutrient solution contained: CaCl2 0,4 mM, KNO3 0,65 mM, MgCl2.6H2O 0,25 mM, (NH4)2SO4 0,01 mM y NH4NO3 0,04 mM. The pH of the solution was adjusted to 4.0 ± 0.1. Nutrient solution was changed daily. Seedling were grown for 4 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Each replicate composed by nine 1 cm long root tips in liquid nitrogen was homogenizated and pre-extracted using Trizol (following Invitrogen’s instructions). Then, EZNA Plant RNA Kit (Omega Bio-tek) was employed for RNA extraction. To remove genomic DNA, a RNAse free DNAse treatment (Invitrogen) was used.
|
| Label |
hy5
|
| Label protocol |
Three biological replicates were independently hybridized for each transcriptomic comparison.Total RNA (1 µg of each) was amplified and aminoallyl-labeled using MessageAmpTM II aRNA kit (Ambion) and 5-(3-aminoallyl)-2’deoxyuridine-5’-triphosphate (aa-dUTP, Ambion) and following the manufacturer´s instructions. For each sample, 3.5 µg of aminoallyl-labeled aRNA was resuspend in 0.1 M Na2CO3 (pH 9.0) and labelled with either Cy3 or Hyper 5 Mono NHS Ester (CyTMDye Post-labelling Reactive Dye Pack, Amersham). The samples were purified following the manufacturer´s instructions for MegaclearTM (Ambion). Cy3 and Hiper 5 incorporation was measured using 1 µl of the probe in a Nanodrop spectrophotometer (Nanodrop Technologies Inc.)
|
| |
|
| Hybridization protocol |
For each hybridization 825 ng of Cy3 and Hiper 5 probes were mixed and was added 11 ul of 10x blocking Agent, 2,2 ul of 25x Fragmentation Buffer and Nuclease free water until 55 ul. The reaction was incubated at 60ºC for exactly 30 minutes to fragment RNA and it was stopped adding the 2xHybridization Buffer. The samples were placed on ice and were loaded onto array as soon as possible. Slides were Agilent White Oligo Microarrays 4x44K (ref. 022297). The arrays was hybridize at 65ºC for 17 hours and then were then washed once for one minute in GE wash buffer 1 at room temperature and once for one minute in GE Wash Buffer 2 at 37ºC. Arrays were drained with a 2000 rpm spin for 2 min
|
| Scan protocol |
Images from Cy3 and Hyper5 channels were equilibrated and captured with a GenePix 4000B (Axon) and spots quantified using GenPix software (Axon)
|
| Description |
Biological replicate 3 of 3. Root apex, addition line 6R, untreated, harvested after 5 days in the growth solution.
0030 wheat 6R replica3 (ct-hy5_exp-cy3) 2-12-10.gpr_Control
raw data file:
0030 wheat 6R replica3 (ct-hy5_exp-cy3) 2-12-10.gpr
|
| Data processing |
Differentially expressed genes were evaluated by the non-parametric algorithm 'Rank Products' available as RankProd package at Bioconductor, an R language project. This method detects genes that are consistently high ranked in a number of replicated experiments independently of their numerical intensities. The results are provided in the form of p-values defined as the probability that a given gene is ranked in the observed position by chance. The expected false discovery rate was controlled to be less than 5%.
Background correction and normalization of expression data were performed using LIMMA. LIMMA is part of Bioconductor, an R language project. For local background correction the normexp method in LIMMA was used. The resulting log-ratios were print-tip loess normalized for each array. To have similar distribution across arrays and to achieve consistency among arrays, log-ratio values were scaled using as scale estimator the median-absolute-value
|
| |
|
| Submission date |
Mar 16, 2017 |
| Last update date |
Mar 17, 2017 |
| Contact name |
Naike Salvador |
| Organization name |
Wake Forest Baptist Medical Center
|
| Street address |
1 Medical Center Blvd
|
| City |
Winston-Salem |
| State/province |
NC |
| ZIP/Postal code |
27103 |
| Country |
USA |
| |
|
| Platform ID |
GPL13627 |
| Series (1) |
| GSE96739 |
Aluminum stress study in wheat and wheat-rye addition lines 3R and 6R. |
|
