|
| Status |
Public on Jan 07, 2014 |
| Title |
NLP6-SUPRD_line7_0hr_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
NLP6-SUPRD line 7 seedling, 0hr, replicate 3
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: whole seedling
age: 3.5-days-old
genotype/variation: 35SΩ-NLP6-NLS-SUPRD transgenic line 7
agent: untreated
|
| Treatment protocol |
After incubation for 3.5 days at 23°C, medium was changed to a fresh medium containing 10 mM KNO3, and after 1 hour of incubation, whole seedlings were collected.
|
| Growth protocol |
Seeds of the parental line (4xNRE-min-GUS line 1) and NLP6-SUPRD lines 7 and 14 were sown into liquid medium containing 2.5 mM ammonium succinate as sole nitrogen source (half-strength Murashige-Skoog medium from which KNO3 and NH4NO3 were omitted, Gamborg's vitamin, 0.5 g/L 2-morpholinoethanesulfonic acid, monohydrate, pH adjusted to 6.5 with KOH, 2.5 mM ammonium succinate). After stratification for 3 days, plates containing the liquid medium and the seeds were transferred to a growth chamber set to a continuous light condition at 23°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Isogen reagent and then treated with DNase I. RNA was purified using the RNeasy mini kit (QIAGEN). The quality of RNA was checked by measuring absorbance at 230, 260 and 280 nm using a spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 1000 ng RNA using the Low-input Quick Amp Labeling Kit, One-Color (Agilent Technologies) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked using a Spectrophotometer.
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| |
|
| Hybridization protocol |
1650 ng 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 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent GE hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent Arabidopsis Gene Expression 4x44k Microarrays (G1529F015241) 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 Technologies) and 1 minute with 37°C GE Wash buffer 2 (Agilent Technologies).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (eXtended Dynamic range scan, Scan Area 61x21.6 mm, Scan resolution 5 um, Dye channel is set to Green and Green PMT is set to XDR Hi 100% and XDR Lo 10%).
|
| Description |
Gene expression before treatment in seedlings of NLP6-SUPRD line 7
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v5_91 and Grid: 021169_D_F_20080807) to obtain background subtracted and spatially detrended Processed Signal intensities.
The 75th percentile signal intensity for each array was calculated using Microsoft Excel software
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| |
|
| Submission date |
Jan 07, 2014 |
| Last update date |
Jan 08, 2014 |
| Contact name |
Shuichi Yanagisawa |
| Organization name |
The University of Tokyo
|
| Department |
Biotechnology Research Center
|
| Street address |
Yayoi 1-1-1
|
| City |
Bunkoy-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
113-8657 |
| Country |
Japan |
| |
|
| Platform ID |
GPL9020 |
| Series (1) |
| GSE53852 |
Effect of the repression of NLP function on nitrate-inducible gene expression in Arabidopsis thaliana |
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