A Multiplex Gene Expression Assay for Direct Measurement of RNA Transcripts in Crude Lysates of the Nematode Caenorhabditis elegans Used as a Bioanalytical Tool
Gene expression profiling in Caenorhabditis elegans has been demonstrated to be a potential bioanalytical tool to detect the toxic potency of environmental contaminants. The RNA transcripts of genes responding to toxic exposure can be used as biomarkers for detecting these toxins. For routine applic...
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description | Gene expression profiling in Caenorhabditis elegans has been demonstrated to be a potential bioanalytical tool to detect the toxic potency of environmental contaminants. The RNA transcripts of genes responding to toxic exposure can be used as biomarkers for detecting these toxins. For routine application in environmental quality monitoring, an easy‐to‐use multiplex assay is required to reliably quantify expression levels of these biomarkers. In the present study, a bead‐based assay was developed to fingerprint gene expression in C. elegans by quantitating messenger RNAs (mRNAs) of multiple target genes directly from crude nematode lysates, circumventing RNA extraction and purification steps. The assay uses signal amplification rather than target amplification for direct measurement of toxin‐induced RNA transcripts. Using a 50‐gene panel, the expression changes of four candidate reference genes and 46 target mRNAs for various contaminants and wastewaters were successfully measured, and the expression profiles indicated the type of toxin present. Moreover, the multiplex assay response was in line with previous results obtained with more time‐consuming reverse‐transcription quantitative polymerase chain reaction and microarray analyses. In addition, the transcriptomic profiles of nematodes exposed to wastewater samples and extracts prepared from tissues of swimming crabs were evaluated. The profiles indicated the presence of organic pollutants. The present study illustrates the successful development of a multiplex fluorescent bead–based approach using nematode C. elegans crude lysates for gene expression profiling of target RNAs. This method can be used to routinely fingerprint the presence of toxic contaminants in environmental samples and to identify the most biologically active fraction of the contaminant mixture in a toxicity identification and evaluation approach. Environ Toxicol Chem 2023;42:130–142. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. |
doi_str_mv | 10.1002/etc.5505 |
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The assay uses signal amplification rather than target amplification for direct measurement of toxin‐induced RNA transcripts. Using a 50‐gene panel, the expression changes of four candidate reference genes and 46 target mRNAs for various contaminants and wastewaters were successfully measured, and the expression profiles indicated the type of toxin present. Moreover, the multiplex assay response was in line with previous results obtained with more time‐consuming reverse‐transcription quantitative polymerase chain reaction and microarray analyses. In addition, the transcriptomic profiles of nematodes exposed to wastewater samples and extracts prepared from tissues of swimming crabs were evaluated. The profiles indicated the presence of organic pollutants. The present study illustrates the successful development of a multiplex fluorescent bead–based approach using nematode C. elegans crude lysates for gene expression profiling of target RNAs. This method can be used to routinely fingerprint the presence of toxic contaminants in environmental samples and to identify the most biologically active fraction of the contaminant mixture in a toxicity identification and evaluation approach. Environ Toxicol Chem 2023;42:130–142. © 2022 The Authors. 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H.</creatorcontrib><creatorcontrib>Dinkla, Inez J. T.</creatorcontrib><creatorcontrib>Murk, Albertinka J.</creatorcontrib><title>A Multiplex Gene Expression Assay for Direct Measurement of RNA Transcripts in Crude Lysates of the Nematode Caenorhabditis elegans Used as a Bioanalytical Tool</title><title>Environmental toxicology and chemistry</title><addtitle>Environ Toxicol Chem</addtitle><description>Gene expression profiling in Caenorhabditis elegans has been demonstrated to be a potential bioanalytical tool to detect the toxic potency of environmental contaminants. The RNA transcripts of genes responding to toxic exposure can be used as biomarkers for detecting these toxins. For routine application in environmental quality monitoring, an easy‐to‐use multiplex assay is required to reliably quantify expression levels of these biomarkers. In the present study, a bead‐based assay was developed to fingerprint gene expression in C. elegans by quantitating messenger RNAs (mRNAs) of multiple target genes directly from crude nematode lysates, circumventing RNA extraction and purification steps. The assay uses signal amplification rather than target amplification for direct measurement of toxin‐induced RNA transcripts. Using a 50‐gene panel, the expression changes of four candidate reference genes and 46 target mRNAs for various contaminants and wastewaters were successfully measured, and the expression profiles indicated the type of toxin present. Moreover, the multiplex assay response was in line with previous results obtained with more time‐consuming reverse‐transcription quantitative polymerase chain reaction and microarray analyses. In addition, the transcriptomic profiles of nematodes exposed to wastewater samples and extracts prepared from tissues of swimming crabs were evaluated. The profiles indicated the presence of organic pollutants. The present study illustrates the successful development of a multiplex fluorescent bead–based approach using nematode C. elegans crude lysates for gene expression profiling of target RNAs. This method can be used to routinely fingerprint the presence of toxic contaminants in environmental samples and to identify the most biologically active fraction of the contaminant mixture in a toxicity identification and evaluation approach. Environ Toxicol Chem 2023;42:130–142. © 2022 The Authors. 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H.</au><au>Dinkla, Inez J. T.</au><au>Murk, Albertinka J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A Multiplex Gene Expression Assay for Direct Measurement of RNA Transcripts in Crude Lysates of the Nematode Caenorhabditis elegans Used as a Bioanalytical Tool</atitle><jtitle>Environmental toxicology and chemistry</jtitle><addtitle>Environ Toxicol Chem</addtitle><date>2023-01</date><risdate>2023</risdate><volume>42</volume><issue>1</issue><spage>130</spage><epage>142</epage><pages>130-142</pages><issn>0730-7268</issn><eissn>1552-8618</eissn><abstract>Gene expression profiling in Caenorhabditis elegans has been demonstrated to be a potential bioanalytical tool to detect the toxic potency of environmental contaminants. The RNA transcripts of genes responding to toxic exposure can be used as biomarkers for detecting these toxins. For routine application in environmental quality monitoring, an easy‐to‐use multiplex assay is required to reliably quantify expression levels of these biomarkers. In the present study, a bead‐based assay was developed to fingerprint gene expression in C. elegans by quantitating messenger RNAs (mRNAs) of multiple target genes directly from crude nematode lysates, circumventing RNA extraction and purification steps. The assay uses signal amplification rather than target amplification for direct measurement of toxin‐induced RNA transcripts. Using a 50‐gene panel, the expression changes of four candidate reference genes and 46 target mRNAs for various contaminants and wastewaters were successfully measured, and the expression profiles indicated the type of toxin present. Moreover, the multiplex assay response was in line with previous results obtained with more time‐consuming reverse‐transcription quantitative polymerase chain reaction and microarray analyses. In addition, the transcriptomic profiles of nematodes exposed to wastewater samples and extracts prepared from tissues of swimming crabs were evaluated. The profiles indicated the presence of organic pollutants. The present study illustrates the successful development of a multiplex fluorescent bead–based approach using nematode C. elegans crude lysates for gene expression profiling of target RNAs. This method can be used to routinely fingerprint the presence of toxic contaminants in environmental samples and to identify the most biologically active fraction of the contaminant mixture in a toxicity identification and evaluation approach. Environ Toxicol Chem 2023;42:130–142. © 2022 The Authors. 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subjects | Amplification Animals Assaying Biological activity Biomarkers Caenorhabditis elegans Caenorhabditis elegans - genetics Contaminants Crustaceans DNA microarrays Environmental monitoring Environmental quality Environmental Toxicology Evaluation Fingerprints Fluorescence Gene expression Gene Expression Profiling Genes Lysates multiplex assay Multiplexing nematode bioassay Nematodes Pollutants Polymerase chain reaction Ribonucleic acid RNA RNA, Messenger - metabolism Swimming Toxicity Toxicology Toxins Transcriptome Transcriptomics Wastewater water quality Water sampling |
title | A Multiplex Gene Expression Assay for Direct Measurement of RNA Transcripts in Crude Lysates of the Nematode Caenorhabditis elegans Used as a Bioanalytical Tool |
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