|
| Status |
Public on May 12, 2011 |
| Title |
TC-pollen-rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Pollen at tricelluar stage, replicate 2
|
| Organism |
Oryza sativa |
| Characteristics |
subspecies: japonica
cultivar: Nipponbare
tissue: pollen
|
| Treatment protocol |
The anthers were fixed in Farmer's fixative (ethanol : acetate = 3 : 1) overnight at 4°C. Dehydration and paraffin embedding were performed by using a microwave processor. Paraffin-embedded sections were cut to a thickness of 16 μm and mounted on PEN membrane glass slides (Molecular Devices, Ontario, Canada) for LM. To remove the paraffin, slides were immersed in 100% xylene (twice), 50% xylene/50% ethanol and 100% ethanol (v/v) for 5 min each, and then air-dried completely at room temperature. Three or four individual flowers were used for each LM experiment. LM was performed using the Veritas Laser Microdissection System LCC1704 (Molecular Devices). Selected areas were captured by an infrared laser (IR laser) onto CapSure Macro LCM Caps (Molecular Devices), and were subsequently cut by a UV laser. The target cells that fused to the LCM cap were collected by removing the cap from the tissue section.
|
| Growth protocol |
Rice (O. sativa L. ssp. japonica cv. Nipponbare) plants were grown in a greenhouse under normal 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. Total RNA was quantified with a Quant-iT™ RiboGreen RNA reagent and kit (Invitrogen, San Diego, CA, USA).
|
| 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). 277 - 1500ng of labeled and fragmented cRNA was hybridized to Agilent Rice 44K custom array (G2519F) 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 pollen at tricelluar stage, collected by lasar microdissection
|
| Data processing |
Scanned tiff image files were analyzed with FeatureExtraction 9.5.1 (Agilent). Obtained background subtracted Signal intensities (gBGSubSignal) were normalized by variance stabilization (VSN) using R software (http://www.r-project.org/).
|
| |
|
| Submission date |
May 11, 2011 |
| Last update date |
May 12, 2011 |
| Contact name |
Koichiro Aya |
| Phone |
81-52-789-5225
|
| Organization name |
Nagoya University
|
| Department |
Bioscience and Biotechnology Center
|
| Lab |
Plant Molecular Breeding
|
| Street address |
Furo-cho, Chikusa-ku
|
| City |
Nagoya |
| State/province |
Aichi |
| ZIP/Postal code |
464-8601 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6864 |
| Series (1) |
| GSE29217 |
Separated Transcriptomes of Male Gametophyte and Tapetum in Rice |
|
