|
| Status |
Public on Apr 25, 2014 |
| Title |
36hr GFP rep B |
| Sample type |
RNA |
| |
|
| Source name |
30 pooled zebrafish embryos
|
| Organism |
Danio rerio |
| Characteristics |
developmental stage: embryo
treatment: injected at the 1-2 cell stage with ~100 pg of enhanced green fluorescent protein (eGFP) IVT mRNA
sampling time: 36 hours post-fertilization (hpf)
replicate: 2
|
| Treatment protocol |
A Narishige IM-300 microinjector was used to microinject 1-2 cell embryos with ~100 pg of dnMTF-1 IVT mRNA (~2.1 nL microinjection volume with a concentration 50 ng/µL IVT mRNA), or ~100 pg of enhanced green fluorescent protein (eGFP) IVT mRNA. The dnMTF-1 is an eGFP fusion protein so proper microinjection and distribution throughout the embryo could be visualized by fluorescent microscopy. Immediately after microinjection, embryos from each treatment were divided into 4 replicates of 30 pooled embryos and held in 25 mL of 0.3X Danieau’s at 28.5°C under a 14 hour light/10 hour dark cycle. eGFP positive embryos (dnMTF-1 and eGFP) were collected at 28 and 36 hpf and flash frozen in liquid nitrogen for future RNA isolation and microarray hybridization.
|
| Growth protocol |
The TL (Tupfel/Long fin mutations) wild-type strain of zebrafish was used for all experiments. Fertilized eggs were obtained from multiple group breedings from a Mass Embryo Production System (Aquatic Habitats, Apopka, FL) with ~200 fish at a ratio of 2 female per 1 male fish. Procedures used in these experiments were approved by the Animal Care and Use Committee of the University of Alabama, Tuscaloosa, Alabama, USA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using the RNA STAT60 protocol (Tel-Test, Inc., TX). RNA was quantified using a Nanodrop 2000C spectrophotometer and assessed for quality using an Agilent 2100 Bioanalyzer Lab-on-chip system. Only samples with RNA integrity number (RIN) between 9.8-10 were used for microarray analysis.
|
| Label |
Cy3
|
| Label protocol |
For each RNA sample, a single microarray was hybridized with 750 ng Cy3 labeled cDNA using Agilent’s standard conditions for single-color microarrays. The Agilent Low-Input QuickAmp Labeling Kit was used for labeling.
|
| |
|
| Hybridization protocol |
The samples were hybridized to a the Agilent 4x44K feature zebrafish microarray using the Agilent In situ Hybridization Kit Plus. Labeled cDNA was combined with the Agilent 10x Control Targets (to identify microarray corners). Microarray hybridizations were performed by Genome Technology Core at the Whitehead Institute for Biomedical Research (Cambridge, MA)
|
| Scan protocol |
Post-hybridization, microarray slides were washed as per the Agilent In situ Hybridization Kit Plus. Arrays were scanned with an Agilent DNA Microarray Scanner.
|
| Description |
30 pooled zebrafish embryos
|
| Data processing |
Extraction of raw microarray results was performed using Agilent’s Feature Extraction software with background detrending (spatial and multiplicative). The data were normalized using a non-linear scaling method based on rank invariant probes. Cy3 signal saturated probes and probes not above background in all replicated were then removed prior to statistical analysis.
|
| |
|
| Submission date |
Sep 30, 2013 |
| Last update date |
Apr 25, 2014 |
| Contact name |
Andrew G. McArthur |
| E-mail(s) |
mcarthua@mcmaster.ca
|
| Organization name |
McMaster University
|
| Department |
Biochemistry & Biomedical Sciences
|
| Street address |
1280 Main Street West
|
| City |
Hamilton |
| State/province |
Ontario |
| ZIP/Postal code |
L8S 4K1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL11077 |
| Series (1) |
| GSE51298 |
Inhibition of endogenous MTF-1 signaling in zebrafish embryos identifies novel roles for MTF-1 in development |
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