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Identification of conserved hepatic transcriptomic responses to 17β-estradiol using high-throughput sequencing in brown trout.

Uren Webster TM, Shears JA, Moore K, Santos EM - Physiol. Genomics (2015)

Bottom Line: Exposure to 34.4 ng E2/L resulted in 2,113 differentially regulated transcripts (FDR < 0.05).We successfully used RNA-Seq to identify highly conserved responses to estrogen and also identified some estrogen-responsive transcripts that have been less well characterized, including nots and tgm2l.These results demonstrate the potential application of RNA-Seq as a valuable tool for assessing mechanistic effects of pollutants in ecologically relevant species for which little genomic information is available.

View Article: PubMed Central - PubMed

Affiliation: Biosciences, College of Life & Environmental Sciences, University of Exeter, Exeter, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Transcript profile analysis for a selection of target genes in all males (A) and females (B), conducted by RT-QPCR. Data are presented as mean fold-change relative to expression in the control group. Relative expression was calculated as ratio of the expression for each target gene/expression for vapa mRNA. Expression of tgm2l was quantified only in mature males and was below the detection limit of the RT-QPCR assay for both immature males and females (n/d, nondetectable). Data were collected from 3–7 males and 2–6 females per treatment group. Individuals for which the expression was below the detection limit of the assay were excluded from the analysis. Asterisks represent significant differences between each treatment group and the control group (*P < 0.05, **P < 0.01, ***P < 0.001).
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Figure 4: Transcript profile analysis for a selection of target genes in all males (A) and females (B), conducted by RT-QPCR. Data are presented as mean fold-change relative to expression in the control group. Relative expression was calculated as ratio of the expression for each target gene/expression for vapa mRNA. Expression of tgm2l was quantified only in mature males and was below the detection limit of the RT-QPCR assay for both immature males and females (n/d, nondetectable). Data were collected from 3–7 males and 2–6 females per treatment group. Individuals for which the expression was below the detection limit of the assay were excluded from the analysis. Asterisks represent significant differences between each treatment group and the control group (*P < 0.05, **P < 0.01, ***P < 0.001).

Mentions: RT-QPCR analysis, performed for eight transcripts using all of the male fish, fully validated the results of the RNA-Seq expression analysis (Fig. 4A). Five upregulated transcripts (vtg1, nots, esr1, zp2.5, zp3a.2) and two downregulated transcripts (tat, tgm2l) were confirmed as being significantly differentially expressed in fish exposed to 34.4 ng/l E2 compared with those in the control group. Furthermore, the expression of esr1 was also significantly increased in both the 1.9 and 18.1 ng/l groups, while zp2.5 and tgm2l were significantly up- and downregulated, respectively, following exposure to 18.1 ng/l, confirming the trends identified in the RNA-Seq dataset. For crot, there was a significant increase in expression of this transcript in male fish exposed to 1.9 ng/l E2 and increasing, but nonsignificant, trends in expression in the two higher treatment groups. RT-QPCR analysis was also performed on the same transcripts in the immature female fish and identified very similar patterns of expression including significant upregulation of vtg1,nots, zp2.5, zp3a.2, crot in the highest treatment group, and significant upregulation of esr1 in fish exposed to both 1.9 and 34.4 ng/l E2 (females were not present in the group of fish exposed to 18.1 ng/l; Fig. 4B). tgm2l expression was detected by RT-QPCR only in sexually mature males (n = 15 across all groups) and not in any immature males (n = 6) or females (n = 10).


Identification of conserved hepatic transcriptomic responses to 17β-estradiol using high-throughput sequencing in brown trout.

Uren Webster TM, Shears JA, Moore K, Santos EM - Physiol. Genomics (2015)

Transcript profile analysis for a selection of target genes in all males (A) and females (B), conducted by RT-QPCR. Data are presented as mean fold-change relative to expression in the control group. Relative expression was calculated as ratio of the expression for each target gene/expression for vapa mRNA. Expression of tgm2l was quantified only in mature males and was below the detection limit of the RT-QPCR assay for both immature males and females (n/d, nondetectable). Data were collected from 3–7 males and 2–6 females per treatment group. Individuals for which the expression was below the detection limit of the assay were excluded from the analysis. Asterisks represent significant differences between each treatment group and the control group (*P < 0.05, **P < 0.01, ***P < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556936&req=5

Figure 4: Transcript profile analysis for a selection of target genes in all males (A) and females (B), conducted by RT-QPCR. Data are presented as mean fold-change relative to expression in the control group. Relative expression was calculated as ratio of the expression for each target gene/expression for vapa mRNA. Expression of tgm2l was quantified only in mature males and was below the detection limit of the RT-QPCR assay for both immature males and females (n/d, nondetectable). Data were collected from 3–7 males and 2–6 females per treatment group. Individuals for which the expression was below the detection limit of the assay were excluded from the analysis. Asterisks represent significant differences between each treatment group and the control group (*P < 0.05, **P < 0.01, ***P < 0.001).
Mentions: RT-QPCR analysis, performed for eight transcripts using all of the male fish, fully validated the results of the RNA-Seq expression analysis (Fig. 4A). Five upregulated transcripts (vtg1, nots, esr1, zp2.5, zp3a.2) and two downregulated transcripts (tat, tgm2l) were confirmed as being significantly differentially expressed in fish exposed to 34.4 ng/l E2 compared with those in the control group. Furthermore, the expression of esr1 was also significantly increased in both the 1.9 and 18.1 ng/l groups, while zp2.5 and tgm2l were significantly up- and downregulated, respectively, following exposure to 18.1 ng/l, confirming the trends identified in the RNA-Seq dataset. For crot, there was a significant increase in expression of this transcript in male fish exposed to 1.9 ng/l E2 and increasing, but nonsignificant, trends in expression in the two higher treatment groups. RT-QPCR analysis was also performed on the same transcripts in the immature female fish and identified very similar patterns of expression including significant upregulation of vtg1,nots, zp2.5, zp3a.2, crot in the highest treatment group, and significant upregulation of esr1 in fish exposed to both 1.9 and 34.4 ng/l E2 (females were not present in the group of fish exposed to 18.1 ng/l; Fig. 4B). tgm2l expression was detected by RT-QPCR only in sexually mature males (n = 15 across all groups) and not in any immature males (n = 6) or females (n = 10).

Bottom Line: Exposure to 34.4 ng E2/L resulted in 2,113 differentially regulated transcripts (FDR < 0.05).We successfully used RNA-Seq to identify highly conserved responses to estrogen and also identified some estrogen-responsive transcripts that have been less well characterized, including nots and tgm2l.These results demonstrate the potential application of RNA-Seq as a valuable tool for assessing mechanistic effects of pollutants in ecologically relevant species for which little genomic information is available.

View Article: PubMed Central - PubMed

Affiliation: Biosciences, College of Life & Environmental Sciences, University of Exeter, Exeter, United Kingdom.

No MeSH data available.


Related in: MedlinePlus