QUANTITATIVE MEASUREMENT OF FATHEAD MINNOW VITELLOGENIN mRNA USING HYBRIDIZATION PROTECTION ASSAYS

Thomas-Jones, Emma; Walkley, Neal A.; Morris, Ceri; Kille, Peter; Cryer, Jennifer; Weeks, Ian; Woodhead, Stuart

doi:10.1897/1551-5028(2003)022<0992:qmofmv>2.0.co;2

Cited by 4 publications

(5 citation statements)

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“…Isolated mRNA was stored in ethanol at −80 °C until analyzed. Liver VTG mRNA was quantified using a VTG mRNA hybridization protection assay (Molecular Light Technology, Cardiff, UK) (see Supporting Information for details).…”

Section: Methodsmentioning

confidence: 99%

Temporal Variation in the Estrogenicity of a Sewage Treatment Plant Effluent and Its Biological Significance

Martinović

Denny

Schmieder

et al. 2007

Environ. Sci. Technol.

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Daily variation in the estrogenic activity of effluent released by a modern sewage treatment plant (STP) was measured and its effects on the physiology, behavior, and reproductive success of male fish were evaluated. As measured by an estrogen receptor binding assay, the daily estrogenic activity of this effluent was both high and extremely variable (42 +/- 25.4 [mean +/- SD] ng 17beta-estradiol (E2) equivalents/L; n = 18). Liver VTG mRNA expression in male fathead minnows (FHM) covaried with the binding assay estimates, suggesting that these fluctuations are biologically relevant. Tests which exposed male FHMs to either fluctuating levels of E2, a constant concentration of E2 (time-weighted to reflect average concentrations), or control (no E2) demonstrated that while the estrogenic activity of this effluent was detrimental to male spawning success, the fact that its concentration varied in a daily manner was without additional influence. The variability of the effluent's estrogenicity suggests that studies concerned with the effects of STP effluents should collect multiple daily samples and then test them on an appropriate time-weighted basis.

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Section: Methodsmentioning

confidence: 99%

Temporal Variation in the Estrogenicity of a Sewage Treatment Plant Effluent and Its Biological Significance

Martinović

Denny

Schmieder

et al. 2007

Environ. Sci. Technol.

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“…; Thomas‐Jones et al . 2003a,b). Three reverse complementary oligonucleotides were designed to the housekeeping gene, beta actin or Fucus VHA‐A sequence with either a C or a U at position 793.…”

Section: Resultsmentioning

confidence: 95%

Intracellular localization and induction of a dynamic RNA‐editing event of macro‐algal V‐ATPase subunit A (VHA‐A) in response to copper

Morris

Owen

Thomas

et al. 2013

Plant Cell & Environment

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A V-ATPase subunit A protein (VHA-A) transcript together with a variant (C793 to U), which introduces a stop codon truncating the subunit immediately downstream of its ATP binding site, was identified within a Fucus vesiculosus cDNA from a heavy metal contaminated site. This is intriguing because the VHA-A subunit is the crucial catalytic subunit responsible for the hydrolysis of ATP that drives ion transport underlying heavy metal detoxification pathways. We employed a chemiluminescent hybridization protection assay to quantify the proportion of both variants directly from mRNA while performing quantification of total transcript using Q-PCR. Polyclonal antisera raised against recombinant VHA-A facilitated simultaneous detection of parent and truncated VHA-A and revealed its cellular and subcellular localization. By exploiting laboratory exposures and samples from an environmental copper gradient, we showed that total VHA-A transcript and protein, together with levels of the truncated variant, were induced by copper. The absence of a genomic sequence representing the truncated variant suggests a RNA editing event causing the production of the truncated VHA-A. Based on these observations, we propose RNA editing as a novel molecular process underpinning VHA trafficking and intracellular sequestration of heavy metals under stress.

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“…To our knowledge, this is the first time the technology has been shown to be capable of quantitatively measuring messenger RNA transcripts that are of medium abundance equivalent to the majority of mRNA transcripts present in the cell. This technology has been used previously with a transcription mediated amplification (TMA) step to measure RNA targets in a qualitative fashion or directly to measure highly abundant transcripts (Thomas-Jones et al, 2003a, 2003bMorris et al, 2014). Teaming this technology with a linear amplification step has a number of consequences; it adds enzymic steps increasing the cost, time to result and observed CVs but it lowers the limit of detection and by doing so, increases the In general, the chemiluminescent assay results reflect the changes determined using Q-PCR though the latter method detected statistically significant gene expression changes in lower doses suggesting it is a more sensitive technique.…”

Section: Amplified Measurement Of Gene Expression Using Chemiluminescmentioning

confidence: 99%

“…In this study, the gene expression of RAD51C, TP53 and CSTA in HEPG2 cells exposed to a range of genotoxic compounds was measured by Q-PCR amplification. In parallel, we developed an alternative assay for the same 3 gene signature based on chemiluminescence (Arnold et al, 1989;Nelson et al, 1996a,b) which we have used for gene expression analysis of abundant genes (Thomas-Jones et al, 2003a, 2003b and the identification of single nucleotide differences in RNA transcripts (Morris et al, 2014). The chemiluminescence method allows the direct quantitation of RNA transcripts by the use of acridinium-ester (AE)-labelled complementary probes and is capable of analytical sensitivity of 10-50 attomol of target with a routine coefficient of variance of less than 15% (Morris et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Quantitative analysis of gene expression changes in response to genotoxic compounds

Morris

El‐Hiti

Weeks

et al. 2017

Toxicology in Vitro

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Techniques that quantify molecular endpoints sufficiently sensitive to identify and classify potentially toxic compounds have wide potential for high-throughput in vitro screening. Expression of three genes, RAD51C, TP53 and cystatin A (CSTA), in HEPG2 cells was measured by Q-PCR amplification. In parallel, we developed alternative assays for the same 3 gene signature based on an acridinium-ester chemiluminescent reporter molecule. HEPG2 cells were challenged with eighteen different compounds (n=18) chosen to represent compounds that are genotoxic (n=8), non-genotoxic non-carcinogenic (n=2) or have a less well defined mechanism of action with respect to genotoxicity (n=8). At least one of the three genes displayed dysregulated expression in the majority of compounds tested by Q-PCR and ten compounds changed the CSTA expression significantly. Acridinium-ester labelled probes for the three genes were synthesised and tested. Analytical sensitivity was characterised and suggested a limit of detection generally better than 0.1fmol but often 10-50 attomol. A linear amplification step was optimised and this quantitative method detected statistically significant increases in RAD51C and CSTA expression in agreement with the Q-PCR results, demonstrating the potential of this technology. The broad agreement of the amplified chemiluminescent method and Q-PCR in measuring gene expression suggests wider potential application for this chemiluminescent technology.

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QUANTITATIVE MEASUREMENT OF FATHEAD MINNOW VITELLOGENIN mRNA USING HYBRIDIZATION PROTECTION ASSAYS

Cited by 4 publications

References 0 publications

Temporal Variation in the Estrogenicity of a Sewage Treatment Plant Effluent and Its Biological Significance

Temporal Variation in the Estrogenicity of a Sewage Treatment Plant Effluent and Its Biological Significance

Intracellular localization and induction of a dynamic RNA‐editing event of macro‐algal V‐ATPase subunit A (VHA‐A) in response to copper

Quantitative analysis of gene expression changes in response to genotoxic compounds

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