|
| Status |
Public on Oct 05, 2012 |
| Title |
XOct-25 overexpression-experiment 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
ectodermal explants isolated at blastula stage and cultured until gastrula stage
|
| Organism |
Xenopus laevis |
| Characteristics |
cell type: embryonic ectodermal cells
expression: XOct-25 overexpressed (dexamethasone treated)
|
| Treatment protocol |
Eight-cell stage embryos were microinjected with 1 ng of mRNA encoding GR-XOct-25, a hormone-inducible version of XOct-25. Ectodermal explants were isolated at stage 9, treated with or without 2 M of dexamethazone (DEX) until the sibling embryos reached stage 10.5, and used for RNA extraction.
|
| Growth protocol |
Xenopus eggs were obtained by artificial fertilization and dejellied with 3.5% cysteine (pH 8.0). Embryos were cultured in 0.5X MMR containing 3.5% Ficoll and staged according to Nieuwkoop and Faber (1967).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from about 100 ectodermal explants was purified using the AGPC method.
|
| Label |
Cy3,Cy5
|
| Label protocol |
Using total RNAs, T7 RNA polymerase-based mRNA amplification was carried out with a RiboAmp RNA Amplification Kit (Arcturus). Five µg of amplified antisense RNA from each sample was labelled with Cy3- or Cy5-dCTP (Cyscribe Post Labeling Reactive Dye; Amersham Biosciences) using random primers and SuperScriptII reverse transcriptase (Invitrogen). Samples were purified using CyScribe GFX Purification Kit (Amersham Biosciences) before hybridization.
|
| |
|
| Channel 2 |
| Source name |
ectodermal explants isolated at blastula stage and cultured until gastrula stage
|
| Organism |
Xenopus laevis |
| Characteristics |
expression: Control (cultured in the absence of dexamethasone )
cell type: embryonic ectodermal cells
|
| Treatment protocol |
Eight-cell stage embryos were microinjected with 1 ng of mRNA encoding GR-XOct-25, a hormone-inducible version of XOct-25. Ectodermal explants were isolated at stage 9, treated with or without 2 M of dexamethazone (DEX) until the sibling embryos reached stage 10.5, and used for RNA extraction.
|
| Growth protocol |
Xenopus eggs were obtained by artificial fertilization and dejellied with 3.5% cysteine (pH 8.0). Embryos were cultured in 0.5X MMR containing 3.5% Ficoll and staged according to Nieuwkoop and Faber (1967).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from about 100 ectodermal explants was purified using the AGPC method.
|
| Label |
Cy5,Cy3
|
| Label protocol |
Using total RNAs, T7 RNA polymerase-based mRNA amplification was carried out with a RiboAmp RNA Amplification Kit (Arcturus). Five µg of amplified antisense RNA from each sample was labelled with Cy3- or Cy5-dCTP (Cyscribe Post Labeling Reactive Dye; Amersham Biosciences) using random primers and SuperScriptII reverse transcriptase (Invitrogen). Samples were purified using CyScribe GFX Purification Kit (Amersham Biosciences) before hybridization.
|
| |
|
| |
| Hybridization protocol |
Microarray hybridization buffer ver2 (Amersham Biosciences) was used for hybridization. Hybridization and washing were carried out according to the manufacturer’s protocol.
|
| Scan protocol |
Scanning was performed on a GenePix 4000B (Axon, Union City, CA) to generate two 16-bit TIFF images corresponding to the Cy3 and Cy5 channels.
|
| Description |
Analysis of the experimental sample overexpressing XOct-25 (dexamethasone treated) in comparison with control sample (dexamethasone non-treated)
|
| Data processing |
After image analysis using GenePix Pro 4.1 (Axon), the data were analysed with the statistical program Cyber-T （http://cybert.microarray.ics.uci.edu/）.
|
| |
|
| Submission date |
Oct 06, 2010 |
| Last update date |
Oct 05, 2012 |
| Contact name |
Atsushi Suzuki |
| Organization name |
Hiroshima University
|
| Department |
Graduate School of Science
|
| Lab |
Institute for Amphibian Biology
|
| Street address |
Kagamiyama 1-3-1
|
| City |
Higashi-hiroshima |
| State/province |
Hiroshima |
| ZIP/Postal code |
739-8526 |
| Country |
Japan |
| |
|
| Platform ID |
GPL11020 |
| Series (1) |
| GSE24562 |
Identification of genes regulated by XOct-25 overexpression in Xenopus ectoderm |
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