Quantification and associated variability of induced vitellogenin gene transcripts in fathead minnow (Pimephales promelas) by quantitative real‐time polymerase chain reaction assay

Abstract

Ecological risk assessors have a growing need for sensitive and rapid indicators of environmental exposures in aquatic ecosystems resulting from natural and synthetic estrogen-like compounds. Investigators developing subcellular exposure markers in traditional sentinel organisms must be vigilant about inherent variability of analyses, especially regarding regulatory and policy statements. Here, we report a quantitative real-time polymerase chain reaction (QPCR) assay for the detection of vitellogenin transcripts environmentally triggered in fathead minnows (Pimephales promelas). We demonstrate that our QPCR assay exhibits little inter- or intra-assay variability (21.7 and 11.9%, respectively). This method appears to be robust in terms of variability stemming from extrinsic sources, indicating that it may be readily transferable to laboratories having the requisite equipment. Our primary focus in development of this method derived from the observation that transcriptional responses of the vitellogenin gene (vtg) in fathead minnows demonstrated high biological variability between identically treated individuals, even under controlled laboratory conditions (coefficient of variation, > 100%). This variability was not seen in other genes from the same RNA preparations that we examined, suggesting that it is specific to the vitellogenin response. Our data and those of others suggest that variability in vtg expression is common to a number of aquatic vertebrates, which is indicative of genetic causation. Despite a relatively high degree of variability in vtg transcription, this method is sensitive enough to detect exposures of 5.0 ng 17alpha-ethinylestradiol (EE2)/L within 24 h of exposure, and it has the ability to discriminate 10.0 and 5.0 ng EE2/L within 48 h. The vitellogenin QPCR assay is a highly sensitive, comparatively rapid, and inexpensive method for the detection and characterization of exposure to environmental estrogens and estrogen mimics.

Collapse of a fish population after exposure to a synthetic estrogen

“…Fish captured for VTG mRNA analysis were euthanized and dissected immediately after capture, and their livers were placed in RNAlater (Ambion, Austin, TX) for transport to the laboratory. Liver VTG mRNA was measured by using quantitative real-time PCR and normalized to 18S ribosomal gene expression, an invariant ribosomal gene, by using methods detailed previously (29).…”

Dynamic Nature of Alterations in the Endocrine System of Fathead Minnows Exposed to the Fungicide Prochloraz

“…All QPCR assays conducted were two-step assays. Samples were extracted, DNAse treated (DNA free; Applied Biosystems/Ambion, Austin, PROCHLORAZ EFFECTS ON FISH ENDOCRINE FUNCTION TX), and then 250 ng total RNA was reverse transcribed to cDNA using methods described by Biales et al (2007). Transcripts for esr1, vtgr, gnrhr1, gnrhr3, cyp19a1b, and cyp51 were quantified using Power SYBR Green PCR Master Mix (Applied Biosystems).…”

Use of gene expression, biochemical and metabolite profiles to enhance exposure and effects assessment of the model androgen 17β‐trenbolone in fish

“…Ovary samples were extracted, DNAse treated (DNAfree; Applied Biosystems/Ambion), and then 250 ng total RNA was reverse-transcribed to complementary DNA, using methods described by Biales et al [14]. Assays were conducted as described previously, using Taqman-based methods for analysis of CYP19A, CYP11A, and AR [15] and a SYBR Green-based method for vitellogenin receptor [15].…”