A quantitative real‐time polymerase chain reaction method for the analysis of vitellogenin transcripts in model and nonmodel fish species

Abstract

The measurement of vitellogenin (vtg) gene transcription has been shown to be a reliable indicator of exposure to estrogenic compounds. Unfortunately, the relatively poor molecular characterization of North American fish species has hindered its application to a larger number of ecologically important species. The current research aimed to demonstrate specific amplification of vtg gene transcripts in three model (zebrafish, rainbow trout, and medaka) and six nonmodel (emerald shiner, pearl dace, smallmouth bass, creek chub, white sucker, and golden redhorse) fish species. Quantitative polymerase chain reaction (QPCR) primers for model species were designed from publicly available vtg sequences. Successful amplification of vtg was demonstrated in fish exposed to 17alpha-ethinylestradiol (EE(2)) for all model species. Vitellogenin primers for selected nonmodel species were designed from published sequences of closely related species. Multiple primers were developed targeting different regions of the vtg gene. The successful amplification of vtg was confirmed through size and sequence analysis for all nonmodel species with the exception of the white sucker, in which amplifications failed. Furthermore, QPCR primers and conditions were quantitative over five orders of magnitude in at least one species (pearl dace) exposed to 5 ng/L of EE(2) for 24 h. The selected species are found in a wide array of ecological habitats that span the United States. Inclusion of vtg transcriptional analysis for wild, ecologically relevant fish in monitoring studies may aid in understanding the extent of estrogenic exposure in aquatic ecosystems across the United States.

Screening complex effluents for estrogenic activity with the T47D‐KBluc cell bioassay: Assay optimization and comparison with in vivo responses in fish

“…All cDNA samples were diluted to 40 ng/ml, and Power SYBR Green PCR Master Mix (Applied Biosystems) was used to determine VTG and ESR1 expression relative to cDNA standards. The QPCRs were conducted as described by Ankley et al [24], using the fathead minnow ESR1 primers (5 0 -CACCCACCAGCCCT-CAG-3 0 /5 0 -CACCTCACACAGACCAACAC-3 0 ) described by Filby and Tyler [22] and the fathead minnow 5 0 region VTG primers (5 0 -CACAATCCCAGCTCTGCGTGA-3 0 /5 0 -TGGCC-TCTGCAGCAATATCAT-3 0 ) developed by Biales et al [25]. Total RNA samples from the effluent exposures on April 13, 2009 andApril 30, 2009, as well as the E2 exposure, were diluted to 10 ng/ml, and VTG and ESR1 relative abundance was determined using Power SYBR Green RNA-to-C T one-step kits (Applied Biosystems), which included RT Enzyme Mix (125 Â ) and Power SYBRGreen RT-PCR Mix (2 Â ).…”

Transcriptional and morphological effects of tamoxifen on the early development of zebrafish (Danio rerio)

“…Vitellogenin is a sensitive indicator of estrogen level, and change in its expression is usually consistent with the environmental estrogen effect (Biales et al ., ; Scholz & Mayer, ; Thorpe et al ., ). When tested with bisphenol A, a significant increase was found even at the lowest test concentration.…”

Affinity and Matrix Effects in Measuring Fish Plasma Vitellogenin Using Immunosorbent Assays: Considerations for Aquatic Toxicologists

“…As a consequence, it is advisable to aquatic toxicologists to avoid comparing absolute calculated plasma vitellogenin concentrations among studies and instead compare either percentages or ratios derived from within the studies to be compared. For example, plasma vitellogenin concentrations in a study can be expressed as % concentration over baseline, as is already widely done when comparing gene mRNA induction [44] or may use a ratio of male vitellogenin in comparison to mean control female plasma vitellogenin, with control female fish assumed to have reproductively optimized plasma vitellogenin concentrations. The latter approach is particularly useful in field studies to compare effects across multiple species (e.g., [45]).…”