A Multiplex Gene Expression Assay for Direct Measurement of RNA Transcripts in Crude Lysates of the Nematode Caenorhabditis elegans Used as a Bioanalytical Tool

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. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Keywords: Caenorhabditis elegans; biomarkers; multiplex assay; nematode bioassay; transcriptomics; water quality.

Figure 1

Comparison between messenger RNA (mRNA) expression measurements by the bead‐based 50‐plex assay, reverse‐transcription quantitative polymerase chain reaction (RT‐qPCR), and microarray assays for a selection of 15 target genes. The regression plots for log2‐transformed mRNA expression data in the nematodes treated with (waste)water are shown. The analysis involved 15 target mRNAs responding to the (waste)water originating from the community, a hospital, a nursing home, and wastewater‐treatment influent or effluent. Pearson correlation coefficients were calculated between the 50‐plex assay and RT‐qPCR (right column) or between the 50‐plex assay and microarray assays (left column). Correlations were considered significant at p < 0.05. FC = fold change; WWTP = wastewater‐treatment plant.

Figure 2

Comparison between messenger RNA (mRNA) expression measurements by the bead‐based 50‐plex assay, reverse‐transcription quantitative polymerase chain reaction (RT‐qPCR), and microarray assays for a selection of nine target genes. The regression plots for log2‐transformed mRNA expression data in nematodes treated with indirect‐acting toxicants are shown. The analysis involved nine target mRNAs of the genes responding to aflatoxin B1, benzo[a]pyrene, or Aroclor 1254. Pearson correlation coefficients were calculated between the 50‐plex assay and RT‐qPCR (right column) or between the 50‐plex assay and microarray assays (left column). Correlations were considered significant at p < 0.05. AFB1 = aflatoxin B1; FC = fold change; B(a)P = benzo[a]pyrene; PCB1254 = Aroclor 1254 (polychlorinated biphenyl 1254).

Figure 3

Transcriptional fingerprint of exposed Caenorhabditis elegans analyzed using a bead‐based 50‐plex gene expression assay. The transcriptional profiles in nematodes exposed to (A) untreated and treated wastewaters, (B) wastewater inflows to wastewater‐treatment plants (WWTPs) from various locations, and (C) organic pollutant extracts from swimming crabs collected in Hangzhou Bay in China. An exposure sample from a nonreceiving surface water was used as control in (A), WWTP effluents related to each of the influent samples tested were used as control in (B), and an exposure sample with 0.5% dimethyl sulfoxide was used as control in (C). The fold change of gene expression level was calculated as the relative messenger RNA amount of a target gene in a test sample and a control sample, normalized to the housekeeping gene tbg‐1 (tubulin gamma chain). Positive values represent up‐regulation; negative values represent down‐regulation. Gene expression levels between 1 and −1 (log2 average fold change) shown in figure by plotted lines were considered as noise. FC = fold change; acdh‐1 = acyl‐coenzyme A dehydrogenase; asp‐13 = aspartyl protease; cdr‐1 = cadmium responsive; cest = carboxyl esterase domain containing; chil‐28 = pseudogene (chitinase like); clec = C‐type lectin; cpt‐3 = carnitine palmitoyl transferase; cyp = cytochrome P450; dhs‐23 = dehydrogenases, short chain; fat‐5 = fatty acid desaturase; gst‐20 = glutathione S‐transferase; lipl‐3 = lipase like; lips‐6 = lipase related; mdh‐1 = malate dehydrogenase; spl‐2 = sphingosine phosphate lyase; tag‐297 = temporarily assigned gene name; ugt = uridine diphosphate glucuronosyltransferase; vmo‐1 = vitelline membrane outer layer 1 homolog; wrt‐4 = warthog.

Figure 3

Transcriptional fingerprint of exposed Caenorhabditis elegans analyzed using a bead‐based 50‐plex gene expression assay. The transcriptional profiles in nematodes exposed to (A) untreated and treated wastewaters, (B) wastewater inflows to wastewater‐treatment plants (WWTPs) from various locations, and (C) organic pollutant extracts from swimming crabs collected in Hangzhou Bay in China. An exposure sample from a nonreceiving surface water was used as control in (A), WWTP effluents related to each of the influent samples tested were used as control in (B), and an exposure sample with 0.5% dimethyl sulfoxide was used as control in (C). The fold change of gene expression level was calculated as the relative messenger RNA amount of a target gene in a test sample and a control sample, normalized to the housekeeping gene tbg‐1 (tubulin gamma chain). Positive values represent up‐regulation; negative values represent down‐regulation. Gene expression levels between 1 and −1 (log2 average fold change) shown in figure by plotted lines were considered as noise. FC = fold change; acdh‐1 = acyl‐coenzyme A dehydrogenase; asp‐13 = aspartyl protease; cdr‐1 = cadmium responsive; cest = carboxyl esterase domain containing; chil‐28 = pseudogene (chitinase like); clec = C‐type lectin; cpt‐3 = carnitine palmitoyl transferase; cyp = cytochrome P450; dhs‐23 = dehydrogenases, short chain; fat‐5 = fatty acid desaturase; gst‐20 = glutathione S‐transferase; lipl‐3 = lipase like; lips‐6 = lipase related; mdh‐1 = malate dehydrogenase; spl‐2 = sphingosine phosphate lyase; tag‐297 = temporarily assigned gene name; ugt = uridine diphosphate glucuronosyltransferase; vmo‐1 = vitelline membrane outer layer 1 homolog; wrt‐4 = warthog.