|
| Status |
Public on Jan 03, 2013 |
| Title |
RiceShoot_NIGT1ox51_2hr_nitrate_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
NIGT1 overexpressor #51 2hr 10 mM KNO3
|
| Organism |
Oryza sativa |
| Characteristics |
organ: shoot
genotype/variation: NIGT1-overexpressing rice (line #51)
treatment: 10 mM nitrate
|
| Treatment protocol |
Rice seedlings were transferred to 10 mM potassium nitrate at 3 hours into the light period and incubated for 2 hours. Shoots were collected.
|
| Growth protocol |
Rice T1 seeds were germinated on agar plates containing 50 ug/ml hygromycin B and grown for 5 days. Resistant seedlings were transferred to a hydroponic setting and grown with distilled water for 9 days in a growth chamber set to 16 hr Light/8 hr Dark conditions at 25｡C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol reagent and then treated with DNase I. After phenol and phenol:chloroform:isoamylalcohol extraction followed by ethanol precipitation, RNA was further 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 and also using the Agilent 2100 Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 500 ng RNA using the One-Color Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked using a Spectrophotometer and the Agilent 2100 Bioanalyzer.
|
| |
|
| Hybridization protocol |
1650 ng of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60｡C for 30 minutes in a reaction volume of 55 ｵl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ｵl of 2x Agilent GE hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent Rice 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 Dymanic 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 in a NIGT1-overexpressing rice (line #51) 2 hours after treatment with 10 mM nitrate
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v5_91 and Grid: 015241_D_F_20110907) to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Jan 02, 2013 |
| Last update date |
Jan 03, 2013 |
| 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 |
GPL8852 |
| Series (1) |
| GSE43243 |
Effect of overexpression of a nitrate-inducible GARP-type transcriptional repressor NIGT1 in rice |
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