|
| Status |
Public on Dec 06, 2012 |
| Title |
NgnrGR plusDex plusGem, rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Ectoderm co-injected with RNAs encoding NgnrGR and geminin, with Ngnr1 activity induced by Dex addition
|
| Organism |
Xenopus laevis |
| Characteristics |
tissue: embryonic ectoderm
rna injection: Dex-inducible version of Ngnr1 (NgnrGR) and geminin
treatment: dexamethasone
|
| Treatment protocol |
When sibling embryos were at stages 10–10.5, explants were pretreated with cycloheximide (final concentration 10mg/ml) for 30min and dexamethasone (final 10 mM) was added to induce transcriptional activity of Ngnr-GR as described previously (EMBO J.26(24):5093-5108). Explants were incubated at 25 degrees C for 2.5 hours and frozen in liquid nitrogen (when sibling embryos were at stage 12.0–12.5).
|
| Growth protocol |
Both blastomeres of two-cell-stage pigmented embryos were injected with X-Ngnr-GR (10pg) with or without geminin (100pg) RNAs (with embryos injected with beta-galactosidase RNA as controls) and were raised until stages 8–9. Xenopus animal caps (50–60 ectodermal explants) per sample were isolated and raised in 0.7MMR at 25 degrees C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared with Trizol (Invitrogen) (Washington University Genome Technology Access Center).
|
| Label |
biotin
|
| Label protocol |
Total RNA (20mg) per sample was used for probe synthesis and hybridization to Affymetrix Xenopus laevis Genome Arrays (Washington University Genome Technology Access Center).
|
| |
|
| Hybridization protocol |
Performed by the Washington University Genome Technololgy Access Center.
|
| Scan protocol |
Affymetrix GeneChip 3000 used by the Washington University Genome Technololgy Access Center.
|
| Data processing |
Result was analyzed with dChip software (http://biosun1.harvard.edu/complab/dchip/). Raw CEL and DAT files were analyzed with dChip software (http://biosun1.harvard.edu/complab/dchip/) after normalization. The entire experiment (microinjection, RNA extraction, and microarray analysis) was repeated two times and quantitative RT-PCR was used to determine cut-offs (see below). Target genes were determined by comparing microarray and quantitative real-time RT-PCR (qRT-PCR) data. Candidates were preliminarily determined from microarray data based on the following criteria: i) expression, represented by model-based expression indices (MBEI), was increased more than 1.2-fold in DEX-treated groups (E/B > 1.2); ii) MBEI differences were larger than 50 (E-B > 50); and iii) expression was claimed as 'present (P) in at least one sample in each experiment. Genes identified as regulated in both experiments were considered candidates. Genes induced in DEX-treated, beta-galactosidase-injected samples compared with the DEX-untreated, Ngnr1-GR-injected samples were also determined and excluded from analysis as they were regarded as DEX-induced. From Ngnr1 candidate target lists, we used qRT-PCR to determine fold-change cut-off values. We found that candidate genes showing E/B > 1.5 and E-B > 70 for Ngnr1 or E/B > 1.3 consistently showed more than 2-fold induction by qRT-PCR.
|
| |
|
| Submission date |
Jul 25, 2012 |
| Last update date |
Jan 23, 2014 |
| Contact name |
Kristen L Kroll |
| E-mail(s) |
kkroll@wustl.edu
|
| Organization name |
Washington University School of Medicine
|
| Department |
Developmental Biology
|
| Lab |
Kristen Kroll
|
| Street address |
320 McDonnell Sciences/660 S. Euclid Ave.
|
| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
| |
|
| Platform ID |
GPL1318 |
| Series (2) |
| GSE39658 |
Regulation of neurogenin-dependent gene expression by geminin |
| GSE39673 |
Geminin regulates the transcriptional and epigenetic status of neuronal fate promoting genes during mammalian neurogenesis |
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