|
| Status |
Public on Mar 31, 2010 |
| Title |
Cy5-isolated cortices & Cy3-whole oocytes |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
whole oocytes
|
| Organism |
Ciona intestinalis |
| Characteristics |
tissue: whole oocytes
|
| Treatment protocol |
Oocytes were dechorionated using either 0.1% trypsin in seawater (pH 8.0) for 3 hours or with 1% thioglycolate, 0.05% pronase in filtered sea water, pH 10.0, for 2-5 minutes (Whole oocytes). Isolated cortices were prepared from oocytes on polylysine-coated coverslip using BufferX as previously described (Prodon et al., 2005, J Cell Sci., 2393-2404).
|
| Growth protocol |
Adult Ciona intestinalis were collected at the Marine Biological Station of Roscoff, France.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Aliquots of total RNA were labeled with either Cy-3 CTP or Cy-5 CTP (Perkin-Elmer/NEN Life Sci.) by two-round linear amplification using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent Tech.) as specified by the manufacture. The quality and size distribution of targets were determined by RNA 6000 Nano Lab-on-chip Assay (Agilent Tech.), and quantification was determined using a NanoDrop microscale spectrophotometer (NanoDrop Technologies).
|
| |
|
| Channel 2 |
| Source name |
isolated cortices
|
| Organism |
Ciona intestinalis |
| Characteristics |
tissue: isolated cortices
|
| Treatment protocol |
Oocytes were dechorionated using either 0.1% trypsin in seawater (pH 8.0) for 3 hours or with 1% thioglycolate, 0.05% pronase in filtered sea water, pH 10.0, for 2-5 minutes (Whole oocytes). Isolated cortices were prepared from oocytes on polylysine-coated coverslip using BufferX as previously described (Prodon et al., 2005, J Cell Sci., 2393-2404).
|
| Growth protocol |
Adult Ciona intestinalis were collected at the Marine Biological Station of Roscoff, France.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
Aliquots of total RNA were labeled with either Cy-3 CTP or Cy-5 CTP (Perkin-Elmer/NEN Life Sci.) by two-round linear amplification using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent Tech.) as specified by the manufacture. The quality and size distribution of targets were determined by RNA 6000 Nano Lab-on-chip Assay (Agilent Tech.), and quantification was determined using a NanoDrop microscale spectrophotometer (NanoDrop Technologies).
|
| |
|
| |
| Hybridization protocol |
A set of 1µg fluorescent-labeled cRNA targets from each sample was assembled into a hybridization reaction on the Ciona intestinalis Oligoarray ver.2, using In Situ Hybridization Kit Plus (Agilent Tech.). Hybridized microarrays were washed according to the manufacture’s protocol.
|
| Scan protocol |
Scanned on an Agilent G2565BA Microarray Scanner System.
|
| Description |
Cy5-isolated cortices & Cy3-whole oocytes
|
| Data processing |
Intensity of probes per array was extracted from scanned microarray images using Feature Extraction software (Agilent Tech. ver.A.7.5.1), which performs background subtraction and dye normalization using the Linear & LOWESS method. All algorithms and parameters used in this analysis were the default condition of the software. The Local Background Subtraction Method was used for background subtractions and the Locally Weighted Linear Regression Method (Linear & LOWESS method) for normalization.
|
| |
|
| Submission date |
Apr 07, 2009 |
| Last update date |
Apr 23, 2009 |
| Contact name |
Lixy Yamada |
| Organization name |
Nagoya University
|
| Department |
Graduate School of Science,
|
| Lab |
Sugashima Marine Biological Laboratory
|
| Street address |
429-63 Sugashima
|
| City |
Toba |
| State/province |
Mie |
| ZIP/Postal code |
517-0004 |
| Country |
Japan |
| |
|
| Platform ID |
GPL5576 |
| Series (1) |
| GSE15586 |
Comparative analysis of gene expression profiles between whole oocytes and isolated cortices |
|
