0 hpf embryos (unfertilized eggs) were collected immediatly after spawning and snap-frozen in dry-ice ethanol bath and stored at -80 oC. MBT embryos were incubated at 28ºC for 3.5 h prior to harvest and snap-freezing.
Growth protocol
The zebrafish was kept in 28 degrees under standard procedures (zebrafish.no).
Extracted molecule
total RNA
Extraction protocol
100 embryos at the time were crushed in a mortar with liquid nitrogen. 2 ml trizol (Triazol reagent fom Invitrogen, CA, U.S.A) was added and the content was transferred to a 15 ml tube and incubated 5 min at RT. 400 μl chloroform was added, embryos were shaken by hand for 15 seconds and further incubated 2-3 min at RT. The lysate was centrifuged (10000 rpm, 10 min, 4ºC), the upper phase was transferred to a new tube, 1 μL glycogen added and RNA was precipitated with 1 mL isopropanol for 10 min at RT before centrifugation (10000 rpm, 10 min, 4ºC) the supernatant was discarded and the pellet washed with 75 % ice-cold ethanol. The samples were then dissolved and treated with DNase and RNeasy (Qiagen; 74104/6).
Label
Cy3
Label protocol
Total RNA (400 ng) and spike-in control RNA was converted to cDNA, followed by cRNA synthesis and amplification, and then purification of cRNA using Agilent's Quick Amp labeling Kit (Product Cat. No: 5190_0442). We followed the protocol from Agilent: One-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling) (Ver.5.7, March 2008; Manual Pat Number:G4140-90040). This method uses T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- labeled CTP which generates cy3 labeled fluorescent cRNA.
Hybridization protocol
Arrays were hybridized (Agilent Hybridization kit, cat. No 5188-5242) for 17 hours (65 °C) and washed before scanning as per the protocol recommended by Agilent (One-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling) (Ver.5.7, March 2008).
Scan protocol
Arrays were scanned using Agilent Technologies Scanner G2505B US45103038 with 5 microns resolution (scan protocol GE1-v5_95_Feb07). Scan area: 61 x 21.6 mm. Grid name: 021643_D_F_20081005. Using the 16 bit tif images generated, feature extraction was performed using Feature Extraction Software 9.5.3 (Agilent Technologies, Inc., CA, USA), with background detrend (FeatNCRange, LoPass) and Multiplicative Detrend.
Description
Gene expression in unfertilized eggs
Data processing
The gProcessed signals from feature extraction were imported into the R-environment (http://cran.r-project.org/). The two groups were normalized separately, using quantile normalization (Bolstad, 2003). The genes of interest were extracted from the dataset. Where there were multiple probes for a single transcript or replicate probes, these were aggregated using the median value (only the genes of interest). These merged intensity values were then log2-transformed and mean and standard deviation were calculated.
