|
| Status |
Public on Jul 30, 2010 |
| Title |
osmpk6 complemented DEX rep3 |
| Sample type |
RNA |
| |
|
| Source name |
osmpk6 complemented line, DEX treatment, 12h, replicate 3
|
| Organism |
Oryza sativa |
| Characteristics |
sample type: suspension cultured cell
genotype: OsMPK6
cultivar: Nipponbare
|
| Treatment protocol |
Suspension cultured cells were transferred to 60 mm dishes (0.4 g cells, 5 ml fresh medium) and maintained in suspension for O.N. at 25˚C with gentle shaking at 100 rpm. Incubations with shaking were continued for various lengths of time after treatments. Dexamethasone (DEX) was used at 10 mM, starting from a 10 mM stock solution in ethanol.
|
| Growth protocol |
Calli of O. sativa L. cv. Nipponbare were suspension-cultured at 25℃ in N6D liquid medium and subcultured in fresh medium every 7days. For the treatment, suspension cell cultures were subcultured for 3 days in a fresh medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the Isogen (Nippongene) and RNeasy Plnat Mini kit (Qiagen) following the manufacturer's recommendations. The protocol includes an on-column DNase digestion. RNA was quantified using a NanoDrop ND-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.4 ug RNA using the Low RNA Input Linear Amplification/Labeling kit (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 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ml of 2x GE buffer was added to the fragmentation mixture and hybridized to Agilent Rice Oligo Microarrays (G2519F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with SSPE Buffer and 1 minute with 37°C SSPE buffer 2, then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides.
|
| Description |
gene expression after 12h in DEX treated cell
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software v9.5 (Agilent) using default parameters (protocol GE1-v5_95_Feb07 and Grid: 015241_D_F_20061020) 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 28, 2009 |
| Last update date |
Jul 30, 2010 |
| Contact name |
Hirohiko Hirochika |
| Organization name |
National Institute of Agrobiological Sciences
|
| Department |
Division of Genome and Biodiversity Research
|
| Street address |
2-1-2 Kannondai
|
| City |
Tsukuba |
| State/province |
Ibaraki |
| ZIP/Postal code |
305-8602 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6864 |
| Series (1) |
| GSE18787 |
Gene regulation in osmpk6 mutant cells expressing OsMKK4DD |
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