|
| Status |
Public on Jul 30, 2018 |
| Title |
root_Fe-deficient_2 |
| Sample type |
RNA |
| |
|
| Source name |
5-day-old wheat seedlings grown for 14 additional days in Fe-deficiency
|
| Organism |
Triticum turgidum subsp. durum |
| Characteristics |
cultivar: Durum wheat (Triticum durum L. cv. Svevo)
developemental stage: 5-day-old wheat seedlings
nutritional condition: Fe-deficiency for 14 additional days
tissue: root
|
| Treatment protocol |
Five-day-old wheat seedlings grown for 14 additional days in four different nutritional conditions (control, C, 1.2 mM sulfate and 80 µM FeIII-EDTA; Fe deficiency, Fe, 1.2 mM sulfate and 0 µM FeIII-EDTA; S deficiency, S, 0 mM sulfate and 80 µM FeIII-EDTA; dual deficiency, D, 0 mM sulfate and 0 µM FeIII-EDTA).
|
| Growth protocol |
Durum wheat seeds (Triticum durum L. cv. Svevo) were germinated for five days in darkness at 20°C on paper towels soaked with distilled water. After germination, uniform seedlings were transferred to plastic pots (12 seedlings per pot) filled with 2.2 l of continuously aerated nutrient solutions. Nutrient solution was changed every 3 days. Plants were grown at 26°C for 14 h of light at 200 µmol m-2 s-1 PAR at leaf level and at 20°C for 10 h in dark (relative humidity 80%). Wheat roots were collected two weeks after the transfer to the NS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the SpectrumTM Plant Total RNA kit (Sigma-Aldrich).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the Low Input Quick Amp Labeling Kit, one-color (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
1.65 µg of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the custom Array 4x44K (GPL13627) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent G2565CA Mycroarray Scanner System (Agilent) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 3 µm, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Fe_2
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (GE1_105_Dec08 and Grid: 022297_D_20160201) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
May 08, 2017 |
| Last update date |
Jul 30, 2018 |
| Contact name |
Anita Zamboni |
| E-mail(s) |
anita.zamboni@univr.it
|
| Phone |
+39 045 8027831
|
| Organization name |
University of Verona
|
| Department |
Biotechnology
|
| Street address |
Strada Le Grazie, 15
|
| City |
Verona |
| ZIP/Postal code |
37134 |
| Country |
Italy |
| |
|
| Platform ID |
GPL13627 |
| Series (1) |
| GSE98634 |
Root transcriptional profile of wheat seedlings in response to Fe, S and dual (Fe and S) deficiencies. |
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