genotype/variation: wild-type AB strain treatment: none
Treatment protocol
Embryos of the appropriate stage, according to development in hpf according to Kimmel et al. (1995), were manually selected under the dissecting scope. Selected embryos were transferred to a basket, rinsed with PBS with 0,7% NaCl, 0,04% triton-X100 and placed on ice in the Trizol solution (GibcoBRL). In the case of embryos at 5 hpf exposed to 10 nM morphine and collected at 24 hpf (covering the complete embryogenesis). Morphine was administered to the embryos in their water environment, i.e., diluted in E3 embryonic medium. The exposition to begun at the stage of 5 hpf (end of blastula) is continuous, in order to study the chronic effects of the exposure to drug.
Growth protocol
Adult zebrafish (wild-type AB strain) were raised in a cycle of 14 h light: 10 h dark at 26ºC in a multi-tank system at our Fish Facilities at the Institute of Neuroscience of Castile & Leon, University of Salamanca. Embryos obtained from natural fertilization were selected at 24 hpf using a Discovery V8 stereomicroscope (Carl Zeiss, Germany), after which fish were raised at 28.5ºC and maintained in dishes containing sterile E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl, 0.33 mM MgSO4) in distilled water.
Extracted molecule
total RNA
Extraction protocol
Total RNA was purified using TRIZOL (Gibco BRL, Gaithersburg, MD, USA) following further RNA purification using an RNeasy Mini Kit for RNA clean-up (Qiagen Sicences, Maryland, USA). RNA quantification and quality was then assessed using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), to test the integrity of the 18S and 28S rRNA bands, and samples with an RNA integrity number (RIN) > 8.0 were used.
Label
biotin
Label protocol
Microarray analysis was performed in the Cancer Research Center (CIC) of Salamanca according to standard procedures. Label protocol was performed according to protocols from Affymetrix. Briefly, 100-300 ng of total RNA were amplified and labeled using the WT Sense Target labelling and control reagents kit (Affymetrix Inc., Santa Clara, CA, USA).
Hybridization protocol
Following fragmentation, cRNA were hybridized to GeneChip® Zebrafish Genome Array (Affymetrix). Washing and scanning were performed using GeneChip System of Affymetrix (GeneChip Hybridization Oven 640, GeneChip Fluidics Station 450 and GeneChip Scanner 7G).
Scan protocol
GeneChips were scanned using the Affymetrix GeneChip Scanner 7G.
Data processing
The RMA (Robust Multi-array Analysis) algorithm was used for background correction and normalization of fluorescent hybridization signals of the microarrays, both at internal (intra-microarrays) and comparative (inter-microarrays) levels. We used Bioconductor and R as computational tools (www.bioconductor.org), to apply RMA to the data set of 12 microarray hybridizations including six different biological replicas corresponding to each of the different experimental groups under study (Control and Morphine). After quantitation of expression level of each probe set in all microarrays analyzed, the SAM algorithm was used to identify probe sets displaying significant differential expression when comparing the treat samples to it controls, using a False Discovery Rate (FDR) of 10% or less as significant.
