|
| Status |
Public on May 06, 2015 |
| Title |
Pax3GR Injected, biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Animal cap explant dissected from embryo injected with Pax3GR mRNA
|
| Organism |
Xenopus laevis |
| Characteristics |
tissue: Blastula stage animal cap explant
treatment: Animal cap explant treated with dexamethasone for 8 hours
injection: Pax3GR
|
| Treatment protocol |
Xenopus laevis embryos were injected at the 2-cell stage with mRNA encoding Pax3GR and Zic1GR , the hormone-inducible version of Pax3 and Zic1, alone or in combination. At the blastula stage (stage9), animal cap explants were dissected and cultured for 8 hours in the presence of dexamethasone. A glucocorticoid receptor (GR) mRNA was also injected as negative control.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Individual animal cap explants were homogenized and total RNA extracted using the RNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions.
|
| Label |
Biotin
|
| Label protocol |
Microarray services were provided by the UPENN Molecular Profiling Facility, including quality control tests of the total RNA samples by Agilent Bioanalyzer and Nanodrop spectrophotometry. All protocols were conducted as described in the NuGEN Ovation User Guide and the Affymetrix GeneChip Expression Analysis Technical Manual. Briefly, 100ng of total RNA was converted to first-strand cDNA using reverse transcriptase primed by poly(T) and random oligomers that incorporated an RNA priming region. Second-strand cDNA synthesis was followed by ribo-SPIA linear amplification of each transcript using an isothermal reaction with RNase, RNA primer and DNA polymerase. The resulting cDNA was fragmented, assessed by Bioanalyzer, and biotinylated by terminal transferase end labeling.
|
| |
|
| Hybridization protocol |
3ug of labeled cDNA was added to Affymetrix hybridization cocktails, heated at 99ºC for 5 min and hybridized for 16 h at 45ºC to Xenopus laevis 2.0 GeneChips (Affymetrix Inc., Santa Clara CA). The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
A GeneChip 3000 7G scanner was used to collect fluorescence signal
|
| Data processing |
Cel files were imported into Partek Genomics Suite (Partek Inc., St. Louis, MO) where GCRMA normalization was applied
|
| |
|
| Submission date |
May 05, 2015 |
| Last update date |
May 06, 2015 |
| Contact name |
Jean-Pierre Saint-Jeannet |
| E-mail(s) |
jsj4@nyu.edu
|
| Phone |
212-998-9978
|
| Organization name |
New York University
|
| Department |
Basic Science and Craniofacial Biology
|
| Street address |
345 East 24th Street
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10010 |
| Country |
USA |
| |
|
| Platform ID |
GPL10756 |
| Series (1) |
| GSE68546 |
Targets of Pax3 and Zic1 in neural crest and cranial placode progenitors |
|
