|
| Status |
Public on Dec 01, 2010 |
| Title |
Ovary_M-_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Ovary without mannitol treatment, replicate 2
|
| Organism |
Oryza sativa |
| Characteristics |
subspecies: japonica
cultivars: Nipponbare
tissue: Ovary
treatment group: without mannitol treatment
|
| Treatment protocol |
To check influences in gene expression of the isolated cells caused by the transient storage in mannitol solution, we prepared RNA samples derived from ovaries with mannitol treatment and another kind of RNA samples derived from ovaries without mannitol treatment, to look for differences in expression between them. Mannitol treatment consisted of soaking five ovaries in mannitol solution for 8 h.
|
| Growth protocol |
Wild-type rice plants (Oryza sativa L. cv. Nipponbare) were grown in pots under natural conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each cell type and tissue with a PicoPure™ RNA isolation kit (Molecular Devices, Ontario, Canada) following the manufacturer's recommendations. The qualities of the RNA samples were assessed using an RNA 6000 Pico kit on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cy3-labeled cRNA were prepared from total RNA using Low RNA Linear Amplification Kit (Agilent) in accordance with the manufacturer's protocol with slight modification. The cDNA synthesis reaction time was extended to 6 hrs.
|
| |
|
| Hybridization protocol |
Fragmentation and hybridization was carried out with Gene Expression Hybridization Kit (Agilent). 400 - 1400ng of labeled and fragmented cRNA was hybridized to microarray using hybridization oven G2545A (Agilent), and wash step was performed with Gene Expression Wash Buffer Kit (Agilent). All operations were performed according to manufacturer's protocol.
|
| Scan protocol |
Microarrays were scanned using Agilent DNA microarray scanner G2565BA according to manufacturer's instructions.
|
| Description |
Gene expression in the rice ovary without mannitol treatment
|
| Data processing |
Scanned tiff image files were analyzed with FeatureExtraction 9.5.1 (Agilent). We slightly modified manufacturer's default extraction protocol called 'GE1-v5_95_Feb07' as follows: 'Background Subtraction Method' was set to 'Average of Negative Control Features', and 'Use Surrogates' was set to 'False'. Then 'gBGSubSignal' columns were extracted from the text data files produced by FeatureExtraction, and introduced into GeneSpring 7.3.1 (Agilent). Positive and negative control features (such as spike-in or dark corner) were removed before data introduction into GeneSpring. Introduced signal intensities were scaled to the 75th percentile per chip (scaled the 75th percentile value was set to 10), and the lowest value of scaled signal intensity was set to 0.01. Microarray intensity values were also normalized per chip by Z score transformation using R software (http://www.r-project.org/).
A scaled data set with the 75th percentile scaling (VALUE) was used in the scatter plot analysis, correlation plot analysis, and to make the lists of the top 10 highly expressed probes. Z-score transformed (per chip) data sets (VALUE2) were also used in the cluster analysis and heat map analysis.
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| |
|
| Submission date |
Mar 25, 2010 |
| Last update date |
Feb 25, 2011 |
| Contact name |
Nori Kurata |
| E-mail(s) |
nkurata@nig.ac.jp
|
| Phone |
+81-55-981-6808
|
| Organization name |
National Institute of Genetics
|
| Lab |
Plant genetics Lab.
|
| Street address |
1111 Yata
|
| City |
Mishima |
| State/province |
Shizuoka |
| ZIP/Postal code |
411-8540 |
| Country |
Japan |
| |
|
| Platform ID |
GPL8852 |
| Series (1) |
| GSE21074 |
Gene expression profiles of the egg and synergid cell in rice |
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