|
| Status |
Public on Dec 01, 2014 |
| Title |
Female control fish 3 at 7d [449901] |
| Sample type |
RNA |
| |
|
| Source name |
Female control, 7d
|
| Organism |
Danio rerio |
| Characteristics |
gender: Female
agent: control
tcdd dose: 0 ppb
time: 7 days
|
| Treatment protocol |
Zebrafish were fed Biodiet starter (4% body weight per day) for 42 d with TCDD added at 0 ppb or 100 ppb (nominal concentration, ethanol used as vehicle).
|
| Growth protocol |
All zebrafish were handled following Animal Care and Use Committee (ACUC) protocols of University of Wisconsin–Milwaukee. Juvenile wild-type zebrafish (stock originally purchased from EkkWill Waterlife Resources, Ruskin, FL) (0.16 g, 2.10 cm) were were reared in 28 °C dechlorinated, filtered municipal water (DFMW) in the NIEHS Children's Environmental Health Sciences Core Center (CEHSC) animal facility.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from individual fish was isolated using Trizol reagent (Invitrogen). One ml of Trizol was used for individual fish, which was homogenized by Power Gen 500 (Fisher), and 0.2 ml of chloroform was added for dissociation of nucleoprotein by shaking the tubes by hand for 15 seconds. The samples were centrifuged at 12000g for 15 minutes at 8℃. Upper aqueous phase was moved to a clean 1.5 ml Eppendorf tube, and 0.5 ml of isopropyl alcohol was added for precipitation of the RNA by incubating the samples at room temperature for 10 minutes. The samples were centrifuged at 12000g for 15 minutes at 8℃. Then the supernatant was removed and 1 ml of 75% ethanol was added for washing the pellet, and the samples were centrifuged at 12000g for 5 minutes at 8℃. The samples were air-dried then dissolved in RNase-free water. The concentration of RNA was determined by ND-1000 NanoDrop Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Further RNA-purification was performed using the RNeasy MiniElute cleanup kit (Qiagen) following the manufacturer’s protocol. Quality of zebrafish RNA was determined by Experion RNA StdSens chip (Bio-Rad).
|
| Label |
Cy3
|
| Label protocol |
RNA of three male and female zebrafish was randomly selected for microarray experiments. cDNA was synthesized using Superscript Double-Stranded cDNA synthesis Kit (Invitrogen). 10ug total RNA of individual fish was denatured at 70℃ for 5min with oligo dT primer, then was reverse transcribed to the first stand cDNA at 42 ℃ for 60 min with SuperScript II enzyme. The second strand cDNA was synthesized at 16℃ with DNA ligase, DNA polymerase I and RNaseH for 2 hours. After digestion with RNase A solution, the double strand DNA was cleaned by Phase Lock tube (Fisher Scientific) with phenol: chloroform: isoamyl alcohol (25:24:1, Ambion), then precipitated in ice-cold ethanol with 7.5M ammonium acetate and 5mg/ml glycogen, dried to the pellet in a DNA 120 SpeedVac (Thermo). Then the DNA was cleaned using Qiaquick PCR purification kit (Qiagen). The quality of DNA was determined by Experion RNA HighSens chip (Bio-Rad). One ug double strand DNA was used for Cy3-labeling reaction with random primer and Klenow fragment (3’->5’ exo-) using One-Color DNA labeling Kit (NimbleGen), Cy3-labeled DNA was precipitated in ice-cold ethanol with 5M NaCl and dried to the pellet in a SpeedVac, then rehydrated in Nuclease-free water. The concentration of Cy3-labeled DNA was determined by the ND-1000 NanoDrop Spectrophotometer.
|
| |
|
| Hybridization protocol |
Cy3-labeled double strand DNA was hybridized to NimbleGen 12X135K array using NimbleGen hybridization kit, the hybridization was performed in NimbleGen Hybridization System (NimbleGen) at 42℃ for 16 hours. Arrays were washed with NimbleGen Wash buffers and 1M DTT, then spinned dry in a NimbleGen microarray drier.
|
| Scan protocol |
The arrays were scanned at 2µm on a NimbleGen MS200 scanner with auto-gain adjust. The TIFF images were gridded and extracted using NimbleScan v. 2.6.
|
| Description |
Female control 3 _WK1
collected at 7 days
|
| Data processing |
Expression data were normalized using the Robust Multichip Average (RMA) algorithm. Differential gene expression were determined with Bayesian Estimation of Temporal Regulation (BETR) by Mev 4.0.
|
| |
|
| Submission date |
May 31, 2013 |
| Last update date |
Oct 11, 2022 |
| Contact name |
Qing Liu |
| E-mail(s) |
liuqingcell@gmail.com
|
| Organization name |
Clemson University
|
| Department |
Biological Sciences
|
| Lab |
Qing Liu
|
| Street address |
Jordan Hall
|
| City |
Clemson |
| State/province |
SC |
| ZIP/Postal code |
29634 |
| Country |
USA |
| |
|
| Platform ID |
GPL17210 |
| Series (2) |
| GSE47547 |
Time-dependent toxic effects of dietary TCDD on juvenile zebrafish |
| GSE47549 |
Toxic effects of dietary TCDD on juvenile zebrafish |
|
