|
| Status |
Public on Sep 23, 2013 |
| Title |
Xenopus embryo_control_rep6 |
| Sample type |
RNA |
| |
|
| Source name |
Xenopus early neurula, uninjected embryo, rep6
|
| Organism |
Xenopus laevis |
| Characteristics |
protocol: uninjected
tissue: whole embryo
developmental stage: Stage 13 (early neurula)
|
| Treatment protocol |
For gain-of-function experiments, 50pg of wild-type Xrx1 mRNA were injected into both dorsal blastomeres of 4-cells stage embryos, using a Drummond ‘Nanoject’ apparatus. For loss-of-function experiments, 10pg of MoXrx1 morpholino antisense oligonucleotide were injeceted into both dorsal blastomeres at 4-cells stage embryos. Embryos were injected in 0.1X MMR and 4% Ficoll-400 (Sigma), and grown overnight at 14°C in the same solution. As experimental controls and for staging purposes, sibling uninjected embryos were grown in 0.1X MMR and 4% Ficoll-400 (Sigma) as well. Embryos for both experiments were collected at stage 13 but an aliquot was kept developing until stage 37/38, to assess final phenotype.
|
| Growth protocol |
After in vitro fertilization and collection. The embryos were cultured in Marc's Modified Ringer (MMR) solution at 0.1X dilution. After half an hour from fertilization, jelly coats were removed in a mildly reducing environment and then rinsed. Embryos were staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
Phycoerithrin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 2 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Xenopus laevis Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400 according to standard Affymetrix protocols.
|
| Scan protocol |
GeneChips were scanned using the GeneChip Scanner 3000 according to standard Affymetrix protocols.
|
| Description |
Labeled sample originates from total RNA pooled from 3 single embryos.
|
| Data processing |
The data were normalized using the GC-RMA algorithm as implemented in the Bioconductor v. 2.9 software package. The data were analyzed using the MeV v. 4.8 software package.
|
| |
|
| Submission date |
Feb 17, 2012 |
| Last update date |
Sep 23, 2013 |
| Contact name |
Massimiliano Andreazzoli |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Pisa
|
| Street address |
SS.12 Abetone e Brennero, 4
|
| City |
Pisa |
| ZIP/Postal code |
56100 |
| Country |
Italy |
| |
|
| Platform ID |
GPL1318 |
| Series (1) |
| GSE35918 |
Expression data of Xrx1 gain and loss of function experiments from early Xenopus laevis embryos (stage 13) |
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