|
| Status |
Public on Oct 01, 2015 |
| Title |
silver_nitrate_induced_replicate_1 |
| Sample type |
RNA |
| |
|
| Source name |
silver nitrate-induced
|
| Organism |
Eutrema salsugineum |
| Characteristics |
stress: silver nitrate
tissue: rosette leaves
|
| Treatment protocol |
For stress treatment leaves were sprayed with 5 mM AgNO3 or placed under a UV lamp (Desaga UVVIS, λ=254 nm, 8 W) at a distance of 20 cm and radiated for 2 h.
|
| Growth protocol |
After 10 days of stratification at 6°C, plants were grown in a growth chamber at a 12/12 h photoperiod at a light intensity of 80 to 100 µmol m-2 s-1 at 21°C and 40% relative humidity.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For each treatment, four biological replicates were investigated, generated from pooled tissues of 4 plants. Total RNA was extracted with NucleoSpin® RNA II Kit (Machery-Nagel). After DNase treatment, concentration and quality of extracted RNA was measured photometrically and with a Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng total RNA using the Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop 2000 Spectrophotometer.
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| |
|
| Hybridization protocol |
0,6 ug of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x GEX Hybridization Buffer HI-RPM was added to the fragmentation mixture and hybridized to a custom Gene Expression Agilent Microarray (G4102A; DesignID 65426) 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).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2600D) using one color scan setting for AgilentG3_GX for 8x60K array slides (Scan Area 61x21.6 mm, Scan resolution 3µm, Dye channel is set to Green and Green PMT is set to 100%, Tiff 20bit).
|
| Description |
Sample name: AgNO3 1
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 11.5.1.1 (Agilent) using default parameters (protocol GE1_1105_Oct12 and Grid: 065426_D_F_20140324) 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 |
Oct 23, 2014 |
| Last update date |
Oct 01, 2015 |
| Contact name |
Erich Glawischnig |
| Organization name |
TU Muenchen
|
| Department |
Plant Science
|
| Street address |
Emil-Ramann-Str. 8
|
| City |
Freising |
| ZIP/Postal code |
85354 |
| Country |
Germany |
| |
|
| Platform ID |
GPL19319 |
| Series (1) |
| GSE62651 |
Substantial Reprogramming of the Thellungiella salsuginea Transcriptome in Response to UV and Silver Nitrate Challenge |
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