|
| Status |
Public on Nov 01, 2012 |
| Title |
Ptf1a-injected embryos NF36, biological rep 3 |
| Sample type |
RNA |
| |
|
| Source name |
Anterior endoderm of 15 Ptf1a-injected embryos NF36
|
| Organism |
Xenopus laevis |
| Characteristics |
developmental stage: NF36
over-expression: GFP+Ptf1a mRNA
tissue: Anterior endoderm
|
| Treatment protocol |
Emrbyos were injected at the 8-cell stage into the two dorsal vegetal blastomeres with either 400 pg of GFP mRNA or 400 pg GFP mRNA and 800 pg Ptf1a mRNA, for dissection tadpoles were anaesthetized in MS-222
|
| 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 |
Gene expression data from 15 Ptf1a-injected embryo, anterior endoderm dissected at NF36
|
| Data processing |
The data were analyzed with Affymetrix expression control using PLIER algorithm and quantile normalization, no scaling was used during the primary analysis.
|
| |
|
| Submission date |
Dec 06, 2011 |
| Last update date |
Nov 01, 2012 |
| Contact name |
Cassandra Kathryn Bilogan |
| E-mail(s) |
cbilogan@mbl.edu
|
| Phone |
508-289-7370
|
| Organization name |
Marine Biology Laboratory
|
| Department |
Eugene Bell Center for Regenerative Biology and Tissue Engineering
|
| Street address |
7 MBL St
|
| City |
Woods Hole |
| State/province |
MA |
| ZIP/Postal code |
02543 |
| Country |
USA |
| |
|
| Platform ID |
GPL10756 |
| Series (1) |
| GSE34193 |
Microarray data Ptf1a gain-of-function Xenopus pancreas development |
|
