NorFluox

Published: 1 December 2021| Version 1 | DOI: 10.17632/42x6pchwhm.1
Contributors:
Laura Guimaraes,
,
,

Description

These data relate to the article entitled "Differential molecular responses of zebrafish larvae to fluoxetine and norfluoxetine" by Rodrigues et al., submitted to Water. Data contained in the file are raw values corresponding to mRNA expression of several zebrafish genes related to mode of action of fluoxetine and its main metabolite. Zebrafish embryos were exposed to concentrations of norfluoxetine or fluoxetine over 80 hpf (hours post-fertilisation). At the end of the exposure, larvae were collected for quantification of mRNA expression by qPCR (SYBRGreen). Details on exposure conditions, gene sequences, primers employed and assay conditions are provided in the submitted article.

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Steps to reproduce

All protocols and details are provided in Rodrigues et al. submitted to Water. Briefly, RNA was extracted using the Illustra RNAspin Mini RNA Isolation kit (GE Healthcare). RNA quality was checked by electrophoresis on an agarose gel of the 18s band and by measuring the optical density ratio at λ260/280nm in a BioTek spectrophotometer. Then, 1µg of total RNA was subjected to digestion of genomic DNA using deoxyribonuclease I Amplification Grade (Invitrogen) and cDNA synthesis was performed using iScript cDNA Synthesis Kit (Biorad). Design of primer pairs was based on gene sequences available in public databases using Primer 3 Plus program. Quantitative real time PCR (qRT-PCR) was employed to assess gene expression. The highest fluorescence signal reached for the lower Cycle threshold (Ct) was used to dictate ideal primer concentrations for qRT-PCR. Primer efficiency was assessed by a series of eight cDNA dilutions ranging from 0.05 to 50ng/μL. The qRT-PCR reactions (10µL of SybrGreen (Biorad), 4µL of water, 2µL of forward primer, 2µl of reverse primer and 2µL of cDNA, in a 20 µL reaction volume) were run in an Eppendorf Mastercycler realplex 4. The reaction parameters were set as follows: 94ºC for 2min; 40 cycles for 30sec at 94ºC for denaturation, for 30sec at respective annealing temperatures, and for another 30 sec at 72ºC for extension; a final extension cycle of 10 minutes at 72ºC was applied. Annealing temperatures were 51ºC for vmat2, 55ºC for 5-ht2c and drdb1, 54ºC for the remaining genes. Blank samples, as well as, melting curves were run for each of the genes assessed (Rodrigues et al. Submitted).

Institutions

Universidade do Porto Centro Interdisciplinar de Investigacao Marinha e Ambiental

Categories

Environmental Toxicology, Biomarkers, Danio Rerio, Antidepressant Medication, Emerging Contaminant

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