|
| Status |
Public on Apr 17, 2009 |
| Title |
Cy5-A4.1+a4.2+b4.2 & Cy3-B4.1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
B4.1 blastomeres
|
| Organism |
Ciona intestinalis |
| Characteristics |
stages: 8-cell stage embryos
|
| Treatment protocol |
Eggs and sperm were obtained surgically from the gonoduct. After insemination, embryos were dechorionated and reared at about 18°C in Millipore-filtered seawater (MFSW) containing 50 μg/ml streptomycin sulfate. Four pairs of blastomere, namely anterior-animal a4.2, posterior-animal b4.2, anterior-vegetal A4.1, and posterior-vegetal B4.1 were isolated with a fine glass needle under a stereomicroscope during 8-cell stage. Isolated blastomeres were collected and treated for RNA extraction before next cell division starts.
|
| Growth protocol |
Adults of Ciona intestinalis were cultivated at the Maizuru Fisheries Research Station of Kyoto University, the Marine Field of Field Science Center.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted from each isolated blastmares 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 |
A4.1+a4.2+b4.2 blastomeres
|
| Organism |
Ciona intestinalis |
| Characteristics |
stages: 8-cell stage embryos
|
| Treatment protocol |
Eggs and sperm were obtained surgically from the gonoduct. After insemination, embryos were dechorionated and reared at about 18°C in Millipore-filtered seawater (MFSW) containing 50 μg/ml streptomycin sulfate. Four pairs of blastomere, namely anterior-animal a4.2, posterior-animal b4.2, anterior-vegetal A4.1, and posterior-vegetal B4.1 were isolated with a fine glass needle under a stereomicroscope during 8-cell stage. Isolated blastomeres were collected and treated for RNA extraction before next cell division starts.
|
| Growth protocol |
Adults of Ciona intestinalis were cultivated at the Maizuru Fisheries Research Station of Kyoto University, the Marine Field of Field Science Center.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted from each isolated blastmares 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.1 (GPL4556), 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 |
none
|
| 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 10, 2009 |
| Last update date |
Apr 13, 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 |
GPL4556 |
| Series (1) |
| GSE15637 |
Comparative analysis of gene expression profiles between four kinds of blastomere at 8-cell stage embryos. |
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