|
| Status |
Public on Apr 01, 2012 |
| Title |
endoderm, NF15, Ngn3GR mRNA, -DEX, biological rep4 |
| Sample type |
RNA |
| |
|
| Source name |
Endoderm of embryos not treated with DEX
|
| Organism |
Xenopus laevis |
| Characteristics |
tissue: endoderm
developmental stage: NF15
overexpression: Ngn3-GR
treatment: no dexamethasone (DEX)
|
| Treatment protocol |
Embryos were cultured in 30 ml of 0.1XMMR in a final concentration of 10 micromolar of dexamethasone.
|
| Growth protocol |
Embryos were grown in 0.1XMMR.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Single-stranded, then double-stranded cDNA was synthesized from the poly(A)+ mRNA present in the isolated total RNA (typically 10 μg total RNA starting material each sample reaction) using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen Corp., Carlsbad, CA ) and poly (T)-nucleotide primers that contained a sequence recognized by T7 RNA polymerase. A portion of the resulting ds cDNA was used as a template to generate biotin-tagged cRNA from an in vitro transcription reaction (IVT), using the Affymetrix GeneChip® IVT Labeling Kit. 15 μg of the resulting biotin-tagged cRNA was fragmented to an average strand length of 100 bases (range 35-200 bases) following prescribed protocols (Affymetrix GeneChip® Expression Analysis Technical Manual).
|
| |
|
| Hybridization protocol |
10 μg of fragmented target cRNA was hybridized at 45°C with rotation for 16 hours (Affymetrix GeneChip® Hybridization Oven 640) to probe sets present on an Affymetrix GeneChip Xenopus Genome array. The GeneChip® arrays were washed and then stained (SAPE, streptavidin-phycoerythrin) on an Affymetrix Fluidics Station 450.
|
| Scan protocol |
The arrays were scanned on a GeneChip® Scanner 3000 7G. The results were quantified and analyzed using GCOS 1.2 software (Affymetrix, Inc.) using default values (Scaling, Target Signal Intensity = 500; Normalization, All Probe Sets; Parameters, all set at default values).
|
| Description |
Xenopus SAMPLE 4 -Dex
Gene expression data from Ngn3-GR injected embryo not treated with DEX. Anterior endoderm dissected at NF15.
|
| Data processing |
The data were analyzed with Flexarray using the RMA algorithm for normalization and the EB(Simon Wright)P algorithm for statistical analysis.
|
| |
|
| Submission date |
May 02, 2011 |
| Last update date |
Apr 01, 2012 |
| Contact name |
Marko Horb |
| E-mail(s) |
mhorb@mbl.edu
|
| Organization name |
Marine Biological Laboratory
|
| Street address |
7 MBL Street
|
| City |
Woods Hole |
| State/province |
MA |
| ZIP/Postal code |
02543 |
| Country |
USA |
| |
|
| Platform ID |
GPL10756 |
| Series (1) |
| GSE29017 |
Expression data from Xenopus endoderm at stage 15 following four hours of Ngn3-GR overexpression |
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