|
Status |
Public on Apr 01, 2012 |
Title |
endoderm, NF15, Ngn3GR mRNA, +DEX, biological rep3 |
Sample type |
RNA |
|
|
Source name |
Endoderm of embryos treated with DEX for four hours at stage 12
|
Organism |
Xenopus laevis |
Characteristics |
tissue: endoderm developmental stage: NF15 overexpression: Ngn3-GR treatment: 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 3 +DEX Gene expression data from Ngn3-GR injected embryo 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 |
|