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The mRNA expression of the octopamine receptor in Balanus improvisus

Poster
Authors Linda Hasselberg
Lena Mårtensson Lindblad
Published in The 14th International Congress on Marine Corrosion and Fouling Abstracts, July 27-31 2008, Kobe - Japan
Pages 60
Publication year 2008
Published at Department of Zoology
Pages 60
Language en
Keywords Real-time PCR, cyprid larvae, barnacle, octopamine receptor
Subject categories Biological Sciences, Cell and molecular biology

Abstract

Medetomidine have been found to strongly inhibit settlement of Balanus improvisus both in the laboratory and in field studies. Our hypothesis is that medetomidine binds to the octopamine receptor, belonging to the family of G-protein coupled receptors. The motive of the study is to investigate if medetomidine may cause tolerance development in barnacles, this due to down-regulation in the mRNA expression of the octopamine receptor. To investigate this possible mechanism for tolerance development, we have developed a protocol for a real-time PCR assay measuring mRNA expression of the octopamine receptor in B. improvisus. Total RNA was isolated both from cyprids and adult animals and RNA from each sample was converted to cDNA using the enzyme reverse transcriptase. To use the real-time PCR, we designed primers to the octopamine receptor sequence to give a PCR product of 69 base pairs. We used the DNA-binding dye SYBR Green, which binds nonspecifically to double-stranded DNA. The fluorescent signal from a reaction is proportional to the amount of double stranded DNA present, and will increase as the target is amplified. The primer concentration was optimized to 200 nM at a temperature of 59°C. The PCR product was sequenced and confirmed to be a part of the octopamine receptor sequence. Primers to a reference gene (actin) have also been designed and tested and we now have an assay for relative quantification with the reference gene as the normalizer. Our aim is to compare the mRNA expression of the octopamine receptor in B. improvisus during different life stages and medetomidine exposures. Financially supported by the Foundation for Strategic Environmental Research (MISTRA), Sweden, through the research program Marine Paint.

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