Optimisation of gene expression analysis in Atlantic salmon lenses by refining sampling strategy and tissue storage

Abstract

Analysis of gene expression in the lens is one of the analytical tools employed to investigate cataract formation in Atlantic salmon (Salmo salar L.). High quality RNA preparations are an essential prerequisite for gene expression analysis. The first aim of the present study was to investigate the possible effects of two methods of tissue preservation on the quality of RNA extracted from Atlantic salmon lenses. RNA was extracted from lenses either stored in RNAlater or flash-frozen in liquid nitrogen. Both tissue preservation methods yielded RNA of similarly high quality. The second aim was to examine if stress related to fish handling and the choice of anaesthesia during the sampling procedure affected gene expression in the lens. Six sampling procedures were tested on groups of sea water adapted Atlantic salmon smolt. Fish were either killed instantaneously (control group) or sampled after 30 min anaesthetised with isoeugenol, after 30 min without anaesthesia, after 120 min anaesthetised with isoeugenol, after 120 min with 15 min anaesthesia with metacaine or after 120 min without anaesthesia. The expression levels of specific genes, of special interest in the study of molecular mechanisms of cataractogenesis, were analysed in lenses by real-time RT-PCR. Fish not anaesthetised had significantly increased levels of heat shock protein 70 (HSP70) mRNA after 30 min compared to the control group. Glutathione reductase (GR) and manganese superoxide dismutase (Mn-SOD) were expressed at significantly lower levels in groups of Atlantic salmon sampled after 120 min anaesthetised with isoeugenol or metacaine, and anaesthetised with isoeugenol, metacaine or without anaesthesia, respectively. The same expression patterns were found in corresponding gill tissues for these two antioxidant genes. In conclusion, preservation in liquid nitrogen instead of RNAlater is recommended due to practical conditions in RNA extraction. A quick sampling protocol with the use of anaesthetics and not exceeding 30 min should be preferred to avoid effects of the sampling procedure on lens gene expression in Atlantic salmon.

Yeast Rhodoturula glutinis as a modulator of innate immune and oxidative stress-related genes in Oreochromis niloticus cultured in a Biofloc system

“…At the end of the experiment, eight fish per treatment were randomly captured and sacrificed according to the AVMA recommendations (Underwood et al 2013) with an overdose of benzocaine. The liver and distal intestine were extracted in cold under aseptic conditions, 50 mg of each tissue stored in RNAlater (Sigma Aldrich, Saint Louis, MI, USA), kept at 4°C overnight, and subsequently stored at -80°C until further processing and analysis (Tröβe et al 2010).…”

Anesthesia for Euthanasia Influences mRNA Expression in Healthy Mice and after Traumatic Brain Injury

“…Data in salmon demonstrate that sedation of 30 min to 2 h before euthanasia and tissue sampling resulted in differential regulation of fast-responding genes. 9,10 Data in rodents are mostly limited to comparison of anesthesia effects during experimental induction of cerebral pathologies. 6,11 There is evidence, however, that the use of isoflurane for euthanasia induces immediate changes in electrophysiology and neuroplasticity compared with decapitation without anesthesia or decapitation with anesthesia 12 ; and that isoflurane anesthesia before CO 2 euthanasia increased c-fos expression in mice brains.…”