tissue: whole body developmental stage: eleuthero-embryo age: 6 days post fertilization treatment: water control
Treatment protocol
All zebrafish embryos used in the experiments were at blastula stage. They were placed in 750 mL covered glass beakers of reconstituted water (total hardness of 125 mg/L as CaCO3 and a conductivity of 270 μS/cm) and the appropriate concentration of PKC412 or DMSO (0.01 %, solvent control). The water temperature was held constant at 27±1 °C with the photoperiod set at 16:8 h light/dark. The static exposure setup consisted of six replicates of water control, solvent control (0.01 % DMSO) and two PKC412 doses. A total of 100 fertilized eggs per replicate (n = 6) were exposed up to 6 days post fertilization (dpf) to nominal concentrations of 2 μg/L and 40 μg/L PKC412, respectively. Every 24 h, lethal and sublethal effects were evaluated, dead embryos or eleuthero-embryos were removed and the water was changed. The quality of the exposure water was continuously monitored by oxygen concentration determination (>70%), the pH value (6.7-7.2) and the temperature (27±1 °C). At the end of exposure, eleuthero-embryos were anaesthetized in a clove oil solution (Fluka AG, Buchs, Switzerland). A total of 80 eleuthero-embryos per replicate were pooled in RNAlater for microarray and qRT-PCR.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from zebrafish eleuthero-embryos using the RNeasy Mini Kit (Qiagen, Basel, Switzerland). Total RNA concentrations were measured spectrophotometrically using a NanoDrop ND-1000 UV-VIS Spectrophotometer at 260 nm. The integrity of each RNA sample was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Basel, Switzerland). Only samples containing a 260/280 nm ratio between 1.8-2.1, a 28S/18S ratio between 1.5-2 and an RNA integrity number (RIN) > 8 were processed further.
Label
Cy3
Label protocol
Total RNA samples (600 ng) were reverse-transcribed into double-strand cDNA in the presence of RNA poly-A controls with the Agilent One-Color RNA Spike-In Kit. Cy3 labeling and hybridization were performed according to the manufacturer's manual. After reverse-transcription of RNA into double-stranded cDNA, double-strand cDNA was in vitro transcribed into cRNA in the presence of Cy3 labeled nucleotides using a Low RNA Input Linear Amp Kit +Cy dye (Agilent Technologies, Basel, Switzerland). The Cy3-labeled cRNA was purified using an RNeasy mini kit (Qiagen, Basel, Switzerland), and quality and quantity was determined using a NanoDrop ND-1000 UV-VIS Spectrophotometer and an Agilent 2100 Bioanalyzer, respectively. Only cRNA samples with a total cRNA yield higher than 2 µg and a dye incorporation rate between 9 pmol/µg and 20 pmol/µg were used for hybridization.
Hybridization protocol
Cy-3-labeled cRNA samples (1.65 µg) were mixed with Agilent blocking solution, subsequently fragmented randomly to 100-200 bp at 65 °C with fragmentation buffer and resuspended in hybridization buffer as provided by the gene expression hybridization Kit (Agilent Technologies). Target cRNA samples (100 µL) were hybridized to the Agilent Zebrafish 4x44K Gene Expression Microarray for 17 h at 65 °C. The hybridized arrays were then washed using Agilent GE wash buffers 1 and 2 according to the manufacturer's instructions.
Scan protocol
Slides were scanned by an Agilent Microarray Scanner (Agilent p/n G2565BA) at 5 μm resolution with the green photomultiplier tube set to 100% and a scan area of 61 x 21.6 mm.
Description
Gene expression after 6 dpf exposure to water control
Data processing
Image generation and feature extraction was performed using the Agilent Feature Extraction (FE) software version 9.5.3. Quality control was additionally considered before performing the statistical analysis. These included array hybridization pattern inspection: absence of scratches, bubbles, areas of non-hybridization, proper grid alignment, spike performance in controls with a linear dynamic range of 5 orders of magnitude and the number of green-feature non-uniformity outliers which should be below 100 for all samples.
Submission date
Jul 26, 2010
Last update date
Jul 23, 2011
Contact name
Daniela Maria Oggier
Organization name
University of Applied Sciences Northwestern Switzerland
