|
| Status |
Public on Dec 31, 2013 |
| Title |
4 dpf-E2-rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Whole embryo, 4 dpf, 1 µM E2 treatment, replicate 1
|
| Organism |
Danio rerio |
| Characteristics |
agent: E2
time: 4 days
developmental stage: 4 dpf embryo
tissue: Whole embryo
|
| Treatment protocol |
17β-estradiol (E2) at a 1 mM stock solution in 100% dimethylsulfoxide (DMSO) was diluted in embryo medium to obtain 1 μM concentrations. A clutch of zebrafish embryos from several pairs of adult fish were divided and transferred into 6 well plates. 30 embryos were pooled as one sample and exposed to 3 ml 1 μM E2 or vehicle (0.1% DMSO) at approximately 3 hours post fertilization (hpf). Embryo media containing E2 were changed every day. At different time points, 1 dpf (24 hpf), 2 dpf (48 hpf), 3 dpf (72 hpf) and 4 dpf (104 hpf), embryos were collected for RNA extractions.
|
| Growth protocol |
Zebrafish embryos were collected after spawning and allowed to develop in a Petri dish in embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) at 28.5˚C with 14:10 LD (14:10 LD; lights on 8 AM; lights off 10 PM.) .
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from pooled embryos was extracted using Trizol (Invitrogen Corporation, Carlsbad, CA) and RNeasy spin columns (Qiagen, Chatsworth, CA) according to the manufacturer’s protocols. On-column DNase I (Qiagen, Chatsworth, CA) digestion was performed to remove remaining DNA. RNA concentrations were measured with NanoDrop 1000 spectrophotometer (Agilent Technologies, Palo Alto, CA) and RNA integrity was analyzed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
|
| Label |
Cy3
|
| Label protocol |
The Agilent Quick Amp Labeling Kit (for one-color) Protocol Version 6.5 was used. The Labeling Kit (Agilent p/n 5190-0442) was used along with Agilent’s RNA Spike-In Kit and Agilent’s Wash Buffers 1 and 2. The RNA Spike-Ins was added to the sample. The sample was simultaneously amplified and Cy3 dye labeled as cRNA was generated using T7 RNA Polymerase. The cRNA was purified using Qiagen RNeasy mini spin columns. Samples were then measured again on the Nanodrop for yield and dye incorporation. Performed by Genomic and RNA Profiling Core, Baylor College of Medicine.
|
| |
|
| Hybridization protocol |
The Agilent’s Hybridization Kit was used for the hybridization. After Cy3 labeling, the samples were fragmented and 1.65 µg of sample and hybridization mix was loaded onto each of the 4x44K Expression arrays. The slide was hybridized in Agilent Hybridization Chamber at 65C at a 10rpm rotation for 17 hours. The slide was washed using the Agilent Expression Wash Buffer Set 1 and 2 as per the Agilent protocol. Performed by Performed by Genomic and RNA Profiling Core, Baylor College of Medicine.
|
| Scan protocol |
The slides were scanned with the Agilent Scanner (G2565BA) using Scanner Version C. Performed by Genomic and RNA Profiling Core, Baylor College of Medicine.
|
| Description |
Gene expression of 4 dpf embryos after 4 days of 1 µM E2 treatment
Sample name: 24984
|
| Data processing |
The Agilent Feature Extraction Software Version 11.0.1.1 was used to analyze the scanned images and processed Signal intensities were obtained after background substraction. Performed by Genomic and RNA Profiling Core, Baylor College of Medicine.
|
| |
|
| Submission date |
Dec 06, 2012 |
| Last update date |
Dec 31, 2013 |
| Contact name |
Ruixin Hao |
| E-mail(s) |
hrx666@hotmail.com
|
| Phone |
7139339425
|
| Organization name |
University of Houston
|
| Department |
Department of biology and biochemistry
|
| Lab |
CNRCS
|
| Street address |
3605 Cullen Blvd, Room 3005
|
| City |
houston |
| State/province |
TX |
| ZIP/Postal code |
77204 |
| Country |
USA |
| |
|
| Platform ID |
GPL6457 |
| Series (1) |
| GSE42766 |
Effect of 17 β-estradiol on developing zebrafish embryos |
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