Selection of reference genes for qRT-PCR examination of wild populations of Atlantic cod Gadus morhua

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Do anesthetics and sampling strategies affect transcription analysis of fish tissues?

<p>Abstract</p> <p>Background</p> <p>The aim of the current examination was to evaluate if sedation and anesthetic treatment techniques affect the quality of RNA extracted from liver, gill, head kidney and brain tissues in Atlantic salmon <it>Salmo salar </it>L. Blood parameters were measured and tissue specimens sampled in six groups of fish; one control group (0 minutes), two groups kept in pure seawater in 90 liter tanks for 30 and 120 minutes, two groups treated with the anesthetic isoeugenol for 30 and 120 minutes, and one group kept in pure seawater for 105 minutes and then anaesthetized with metacaine for 15 minutes. RNA quality was assessed with the NanoDrop ND-1000 spectrophotometer (260/280 and 260/230 nm ratios) and with the Agilent Bioanalyzer (28S/18S ratio and RIN data) in samples either preserved in liquefied nitrogen (N₂) or in RNA<it>later</it>. In addition, the transcriptional levels of two fast-responding genes were quantified in gill and brain tissues.</p> <p>Results</p> <p>The results show that physiological stress during sampling does not affect the quality of RNA extracted from fish specimens. However, prolonged sedation (2 hours) resulted in a metabolic alkalosis that again affected the transcriptional levels of genes involved in ionoregulation and respiration. In gills, <it>Na</it>⁺-<it>K</it>⁺-<it>ATPase α1b </it>was significantly downregulated and <it>hypoxia inducible factor 1 </it>(<it>HIF1</it>) significantly upregulated after two hours of treatment with isoeugenol, suggesting that this commonly used sedative affects osmo-regulation and respiration in the fish. The results also suggest that for tissue preservation in general it is better to flash-freeze fish specimens in liquefied N₂than to use RNA<it>later</it>.</p> <p>Conclusion</p> <p>Prolonged sedation may affect the transcription of fast-responding genes in tissues of fish. Two hours of sedation with isoeugenol resulted in downregulation of the <it>Na</it>⁺-<it>K</it>⁺-<it>ATPase α1b </it>gene and upregulation of the <it>HIF1 </it>gene in gills of Atlantic salmon. The quality of RNA extracted from tissue specimens, however, was not affected by sedation treatment. Flash-freezing of tissue specimens seems to be the preferred preservation technique, when sampling fish tissue specimens for RNA extraction.</p

Spatial transcription of CYP1A in fish liver

<p>Abstract</p> <p>Background</p> <p>The aim of this work was to study how evenly detoxifying genes are transcribed spatially in liver tissue of fish. Ten Atlantic salmon <it>Salmo salar </it>were intraperitoneally injected with 50 mg/kg of the strong CYP1A inducer β-naphthoflavone and liver tissue harvested seven days later. The liver from 10 control and 10 exposed fish were split into eight sections, RNA extracted and three reference (β-actin, elongation factor 1A_B(EF1A_B)) and two detoxifying genes (CYP1A and GST) quantified with real-time RT-PCR. The cellular localization of the EF1A_Band CYP1A mRNA in the liver of control and β-naphthoflavone treated fish was then determined by <it>in situ </it>hybridization (ISH) using EF1A_Band CYP1A biotinylated oligonucleotide probes.</p> <p>Results</p> <p>The study shows that genes encoding phase I and phase II conjugating enzymes are unevenly transcribed in different parts of the liver of Atlantic salmon seven days after a single-dose of β-naphthoflavone exposure. Transcription of CYP1A and GST was higher in the middle section of the liver compared to the distal and proximal parts of the organ. The ISH data suggest that CYP1A transcription happens mainly in hepatocyte cells in the liver, and that hepatocytes in the vicinity of blood vessels respond stronger to β-naphthoflavone than cells further away from the blood supply.</p> <p>Conclusion</p> <p>Overall, the qRT-PCR and ISH results reported here suggest that gene expression analysis should be performed on as pure cell populations as possible. If bulk tissue samples are to be used, one should always check how evenly the target genes are expressed in tissue sections and organs in every study.</p

Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon

BACKGROUND: Salmonid fishes are among the most widely studied model fish species but reports on systematic evaluation of reference genes in qRT-PCR studies is lacking. RESULTS: The stability of six potential reference genes was examined in eight tissues of Atlantic salmon (Salmo salar), to determine the most suitable genes to be used in quantitative real-time RT-PCR analyses. The relative transcription levels of genes encoding 18S rRNA, S20 ribosomal protein, β-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), and two paralog genes encoding elongation factor 1A (EF1A(A )and EF1A(B)) were quantified in gills, liver, head kidney, spleen, thymus, brain, muscle, and posterior intestine in six untreated adult fish, in addition to a group of individuals that went through smoltification. Based on calculations performed with the geNorm VBA applet, which determines the most stable genes from a set of tested genes in a given cDNA sample, the ranking of the examined genes in adult Atlantic salmon was EF1A(B)>EF1A(A)>β-actin>18S rRNA>S20>GAPDH. When the same calculations were done on a total of 24 individuals from four stages in the smoltification process (presmolt, smolt, smoltified seawater and desmoltified freshwater), the gene ranking was EF1A(B)>EF1A(A)>S20>β-actin>18S rRNA>GAPDH. CONCLUSION: Overall, this work suggests that the EF1A(A )and EF1A(B )genes can be useful as reference genes in qRT-PCR examination of gene expression in the Atlantic salmon

A data driven approach to landslide susceptibility mapping in Great Britain

Landslides are a geo-hazard which can have significant societal impacts including loss of human life, physical damage to infrastructure and financial loss. The ability to assess where landslides will occur is therefore of great interest for the public good and can be approached both theoretically and empirically. With the ever increasing availability of spatial data, information on landslide events is now much more readily available ranging from initiation point coordinates to high (sub-metre) resolution topographic information and associated derivatives on affected (and unaffected) areas. Coupled with information on the geology of a region, it is possible to build up a detailed location specific profile of past events, all of which may prove useful for informing where future events may occur. We present preliminary results from an assessment of various data to reassess current British landslide susceptibility datasets. These could be used in future to provide additional information to support landslide forecasting. We define susceptibility as: The potential for the occurrence of a hazard within a specified area. This is currently provided for by the BGS GeoSure Landslides product [1] which classifies landslide prone areas on an A-E (low-high) basis, based on heuristics as well as consideration of lithology, discontinuities and slope angle. Data-driven analyses may provide further insights into where and why landslides occur. Using this knowledge, we hope to improve our current landslide susceptibility model. Consequently, this will enable us to be more confident in the identification of areas where landslides may occur in the future

Ontogeny-Specific Skeletal Deformities in Atlantic Haddock Caused by Larval Oil Exposure

Bone deformities are one of the main effects of crude oil exposure in marine fish larvae. Craniofacial and jaw deformities, if severe enough, may restrict feeding and ultimately kill the developing larvae. This study aimed to examine the impact of dispersed crude oil on bone development in Atlantic haddock (Melanogrammus aeglefinus) larvae, a fish species spawning in areas approached for oil and gas exploration in the North Atlantic Ocean. Atlantic haddock larvae were exposed to low (60 μg oil/L), high (600 μg oil/L), or pulsed (0–600, average 60 μg oil/L over time) dispersed crude oil from 0 to 18 days post hatch (dph). Endpoints included survival and growth, bone integrity, and transcriptional parameters, which were assessed during (0–18 dph) and after exposure until the fish reached 8 months of age (243 dph). The results showed that the larvae in the high treatment group had reduction in growth at 2–19, 44, 134, and 243 dph. Craniofacial abnormalities were most severe at 8 and 19 dph. These deformities were not present at 44 dph, possibly because the larvae with deformed jaws failed to feed properly and died. Higher prevalence of spinal deformities was observed in haddocks that survived for 243 dph. Three genes encoding proteins critical for osteoblast function, sp7, postn, and col10a1, were downregulated in the high treatment group larvae. We discuss possible mechanisms of action in the developing larvae after oil exposure. In conclusion, this study shows that larval exposure to oil can potentially have long-term effects on growth and bone integrity in Atlantic haddock.publishedVersio

Pharmacokinetics and transcriptional effects of the anti-salmon lice drug emamectin benzoate in Atlantic salmon (Salmo salar L.)

Background Emamectin benzoate (EB) is a dominating pharmaceutical drug used for the treatment and control of infections by sea lice (Lepeophtheirus salmonis) on Atlantic salmon (Salmo salar L). Fish with an initial mean weight of 132 g were experimentally medicated by a standard seven-day EB treatment, and the concentrations of drug in liver, muscle and skin were examined. To investigate how EB affects Atlantic salmon transcription in liver, tissues were assessed by microarray and qPCR at 7, 14 and 35 days after the initiation of medication. Results The pharmacokinetic examination revealed highest EB concentrations in all three tissues at day 14, seven days after the end of the medication period. Only modest effects were seen on the transcriptional levels in liver, with small fold-change alterations in transcription throughout the experimental period. Gene set enrichment analysis (GSEA) indicated that EB treatment induced oxidative stress at day 7 and inflammation at day 14. The qPCR examinations showed that medication by EB significantly increased the transcription of both HSP70 and glutathione-S-transferase (GST) in liver during a period of 35 days, compared to un-treated fish, possibly via activation of enzymes involved in phase II conjugation of metabolism in the liver. Conclusion This study has shown that a standard seven-day EB treatment has only a modest effect on the transcription of genes in liver of Atlantic salmon. Based on GSEA, the medication seems to have produced a temporary oxidative stress response that might have affected protein stability and folding, followed by a secondary inflammatory response.publishedVersio

Is chemically dispersed oil more toxic to Atlantic cod (Gadus morhua) larvae than mechanically dispersed oil? A transcriptional evaluation

Abstract Background The use of dispersants can be an effective way to deal with acute oil spills to limit environmental damage, however very little is known about whether chemically dispersed oil have the same toxic effect on marine organisms as mechanically dispersed oil. We exposed Atlantic cod larvae to chemically and mechanically dispersed oil for four days during the first-feeding stage of development, and collected larvae at 14 days post hatch for transcriptional analysis. A genome-wide microarray was used to screen for effects and to assess whether molecular responses to chemically and mechanically dispersed oil were similar, given the same exposure to oil (droplet distribution and concentration) with and without the addition of a chemical dispersant (Dasic NS). Results Mechanically dispersed oil induced expression changes in almost three times as many transcripts compared to chemically dispersed oil (fold change >+/−1.5). Functional analyses suggest that chemically dispersed oil affects partly different pathways than mechanically dispersed oil. By comparing the alteration in gene transcription in cod larvae exposed to the highest concentrations of either chemically or mechanically dispersed oil directly, the chemically dispersed oil affected transcription of genes involved nucleosome regulation, i.e. genes encoding proteins participating in DNA replication and chromatin formation and regulation of cell proliferation, whereas the mechanically dispersed oil most strongly affected genes encoding proteins involved in proteasome-mediated protein degradation. Cyp1a was the transcript that was most strongly affected in both exposure groups, with a 60-fold induction in the two high-exposure groups according to the RT-qPCR data, but no significant difference in transcriptional levels was observed between the two treatments. Conclusions In summary, dispersants do not appear to add to the magnitude of transcriptional responses of oil compounds but rather appear to lower or modify the transcriptional effect on cod larvae.</p