|
| Status |
Public on Oct 27, 2016 |
| Title |
Arabidopsis_ITC-0.05M_9h_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Rosette leaves, 10 days, 0.05M ITC, 9h
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col-0
|
| Treatment protocol |
Allyl-isothiocyanate (Sigma-Aldrich, Saint Louis, USA; Cat Nb 377430) was freshly diluted in commercial rape seed oil to a concentration of 0.05M and 200 µl of this solution was applied to a piece of filter paper that was placed into a 14-cm diameter Petri dish. Exposure to allyl-isothiocyanate was obtained by putting a 9-cm dish (lid removed) containing ten-day old A. thaliana plants into this 14-cm diameter dish for 30 minutes, 1 hour or 9 hours. The plants were hence exposed to vapors of allyl-isothiocyanate in a closed atmosphere. The control consisted of filter paper onto which 200 µl of rape seed oil was applied.
|
| Growth protocol |
Seeds of the A. thaliana accession Col-0 were surface sterilised and sown on Petri dishes (9 cm diameter) containing solid in vitro cultivation medium consisting of ½ x Murashige and Skoog basal salt mixture (Sigma-Aldrich, Saint Louis, USA), 2% sucrose, 0.6% phytoagar (w/v), pH 5.7. Seeds were stratified for 2 days at 4°C before being transferred to a controlled growth chamber under a 16 hour photoperiod (light intensity: 75 µmol.m-2.sec-1) at 21-23°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Shoots from 10 days old in vitro grown plantlets were harvested separately from two individual petri dishes and immediately flash-frozen in liquid N2. The harvested tissue was stored at -80°C until further processing. Precooled (-80°C) 5 mm stainless steel beads (QIAGEN) were added to the tubes with frozen plant tissue, and the samples were mechanically disrupted and homogenized using the TissueLyser system (QIAGEN). Disruption was carried out for 2 x 2 min at 25 Hz. The samples were placed in a precooled (-80°C) adapter set for the first shaking step. Before the second shaking step, the samples were transferred to a room temperate adapter set and 0.5 ml lysis buffer (SpectrumTM Plant Total RNA kit, Sigma-Aldrich) was added to each tube. Total RNA was isolated from the homogenized lysate using the SpectrumTM Plant Total RNA kit (Sigma-Aldrich). On-column digestion of DNA with DNase I (QIAGEN) was included for all RNA preparations. The concentration of the RNA was determined by measuring the absorbance at 260 nm with the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies), and the purity of the RNA was estimated by the OD260/OD280 nm absorption ratio (all ratios were > 2.0). The integrity of the RNA was verified by denaturing agarose gel electrophoresis and ethidium bromide staining following the protocol described in the RNeasy Mini handbook (QIAGEN).
|
| Label |
Cy3
|
| Label protocol |
Total RNA (83 - 200 ng) was reverse transcribed, amplified and labelled with Cy3-CTP using the Low Input Quick Amp Labelling Kit, One-Color (Agilent p/n 5190-2305)
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| |
|
| Hybridization protocol |
1650 ng cRNA from each sample was fragmented and hybridized on 4x44K Arabidipsis thaliana whole-genome 60-mer oligonucleotide microarrays (Agilent Technologies) in an Agilent G2545A Hybridization rotary oven (10 rpm, 65°C, 17.5 h). Hybridization was performed with the Gene Expression Hybridization Kit (Agilent p/n 5188-5242). The slides were washed with buffer 1 & 2 from Gene Expression Wash Buffer kit (Agilent p/n 5188-5327)
|
| Scan protocol |
The microarray sides were scanned twice at 5 µm resolution on a laser scanner (G2505 B from Agilent Technologies), using the “dynamic range expander” option in the scanner software. The resulting images were processed using Agilent Feature Extraction software v9.5.
|
| Data processing |
The median signal intensities (gMedianSignal) were normalized using the quantile method, and no background subtraction was performed. Spots identified as feature outliers were excluded from analysis, and weak or non-detected spots were given reduced weight (0.5). Genes with statistically significant differential expression were identified using the Limma package (version 3.2.3) and R (version 2.10.1).
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| |
|
| Submission date |
May 19, 2016 |
| Last update date |
Oct 27, 2016 |
| Contact name |
Per Winge |
| E-mail(s) |
per.winge@ntnu.no
|
| Phone |
+47 73596229
|
| Organization name |
Norwegian University of Science and Technology
|
| Department |
Department of Biology
|
| Lab |
Bones lab
|
| Street address |
Høgskoleringen 5e
|
| City |
Trondheim |
| ZIP/Postal code |
7491 |
| Country |
Norway |
| |
|
| Platform ID |
GPL12621 |
| Series (1) |
| GSE81634 |
Allyl-isothiocyanate treatment induces a complex transcriptional reprogramming including heat stress, oxidative stress and plant defense responses in Arabidopsis thaliana |
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