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Spatial transcription of CYP1A in fish liver.

Olsvik PA, Lie KK, Saele Ø, Sanden M - BMC Physiol. (2007)

Bottom Line: Transcription of CYP1A and GST was higher in the middle section of the liver compared to the distal and proximal parts of the organ.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Nutrition and Seafood Research, N-5817 Bergen, Norway. pal.olsvik@nifes.no

ABSTRACT

Background: The aim of this work was to study how evenly detoxifying genes are transcribed spatially in liver tissue of fish. Ten Atlantic salmon Salmo salar were intraperitoneally injected with 50 mg/kg of the strong CYP1A inducer beta-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 (beta-actin, elongation factor 1AB (EF1AB)) and two detoxifying genes (CYP1A and GST) quantified with real-time RT-PCR. The cellular localization of the EF1AB and CYP1A mRNA in the liver of control and beta-naphthoflavone treated fish was then determined by in situ hybridization (ISH) using EF1AB and CYP1A biotinylated oligonucleotide probes.

Results: 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 beta-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 beta-naphthoflavone than cells further away from the blood supply.

Conclusion: 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.

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Related in: MedlinePlus

Reference gene stability in eight sections of Atlantic salmon Salmo salar liver. Y-axis = quantities transformed from raw Ct values. ● = exposed, □ = control.
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Figure 4: Reference gene stability in eight sections of Atlantic salmon Salmo salar liver. Y-axis = quantities transformed from raw Ct values. ● = exposed, □ = control.

Mentions: In this work, the transcription levels of three reference genes were quantified, and a normalization factor calculated by the geNorm software used to quantify mean normalized expression of CYP1A and GST. In liver tissue, β-actin was the most stable reference gene, whereas EF1AB and ARP were the most stable reference genes in gills and head kidney, respectively. These stability measurements were based on the M value, as calculated by the geNorm software. Figure 4 shows the stability of each the three reference genes as quantities across the eight sections of liver. The highest relative quantities for each gene are set to 1. These raw, not yet normalized, reference gene quantities are the required data input for geNorm. The quantities are not saying anything about up- or downregulation per se. They can, however, be used to visualize reference gene stability. Based on the quantities, these results indicate that EF1AB (Fig. 4B) was stable within both the control and exposed groups, and that ARP (Fig. 4C) seems to be downregulated in the exposed group as compared to the control group. But since the stability of ARP was uniform within the control and exposed groups (Kruskal-Wallis, Control P = 0.99, Exposed P = 0.98), this gene can still be used in calculations of the normalization factor in examinations of spatial target gene expression throughout the liver.


Spatial transcription of CYP1A in fish liver.

Olsvik PA, Lie KK, Saele Ø, Sanden M - BMC Physiol. (2007)

Reference gene stability in eight sections of Atlantic salmon Salmo salar liver. Y-axis = quantities transformed from raw Ct values. ● = exposed, □ = control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2238752&req=5

Figure 4: Reference gene stability in eight sections of Atlantic salmon Salmo salar liver. Y-axis = quantities transformed from raw Ct values. ● = exposed, □ = control.
Mentions: In this work, the transcription levels of three reference genes were quantified, and a normalization factor calculated by the geNorm software used to quantify mean normalized expression of CYP1A and GST. In liver tissue, β-actin was the most stable reference gene, whereas EF1AB and ARP were the most stable reference genes in gills and head kidney, respectively. These stability measurements were based on the M value, as calculated by the geNorm software. Figure 4 shows the stability of each the three reference genes as quantities across the eight sections of liver. The highest relative quantities for each gene are set to 1. These raw, not yet normalized, reference gene quantities are the required data input for geNorm. The quantities are not saying anything about up- or downregulation per se. They can, however, be used to visualize reference gene stability. Based on the quantities, these results indicate that EF1AB (Fig. 4B) was stable within both the control and exposed groups, and that ARP (Fig. 4C) seems to be downregulated in the exposed group as compared to the control group. But since the stability of ARP was uniform within the control and exposed groups (Kruskal-Wallis, Control P = 0.99, Exposed P = 0.98), this gene can still be used in calculations of the normalization factor in examinations of spatial target gene expression throughout the liver.

Bottom Line: Transcription of CYP1A and GST was higher in the middle section of the liver compared to the distal and proximal parts of the organ.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Nutrition and Seafood Research, N-5817 Bergen, Norway. pal.olsvik@nifes.no

ABSTRACT

Background: The aim of this work was to study how evenly detoxifying genes are transcribed spatially in liver tissue of fish. Ten Atlantic salmon Salmo salar were intraperitoneally injected with 50 mg/kg of the strong CYP1A inducer beta-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 (beta-actin, elongation factor 1AB (EF1AB)) and two detoxifying genes (CYP1A and GST) quantified with real-time RT-PCR. The cellular localization of the EF1AB and CYP1A mRNA in the liver of control and beta-naphthoflavone treated fish was then determined by in situ hybridization (ISH) using EF1AB and CYP1A biotinylated oligonucleotide probes.

Results: 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 beta-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 beta-naphthoflavone than cells further away from the blood supply.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus