A Whole-Embryo RT-qPCR Method for Measuring Human Gene Expression in Zebrafish Xenografts
Vagg, Sydney
Vagg, Sydney
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Abstract
The zebrafish xenograft model is widely used in cancer research due to its rapid development, transparent embryos, and conserved genetic pathways with humans. In this model, human tumor cells interact with the zebrafish vasculature and are exposed to physiological stresses such as fluid shear stress, which can influence cancer cell survival and transcriptional changes. Understanding how circulating tumor cells (CTCs) respond to vascular stress may help identify mechanisms that allow metastatic cells to survive in circulation. The goal of this project is to develop a method to measure transcriptional changes in human tumor cells within zebrafish embryos. Traditional approaches require dissociating embryos and sorting GFP-positive human cells, which is labor intensive. Instead, we aim to establish a reverse transcription quantitative PCR (RT-qPCR) method that detects human transcripts directly from RNA extracted from whole zebrafish embryos. To develop this method, we identified human-specific housekeeping gene primers that would not amplify zebrafish transcripts. Two candidates, HPRT1 (hypoxanthine phosphoribosyltransferase 1) and TBP (TATA-binding protein), were selected from previous studies. RT-PCR using RNA from zebrafish embryos and human lung cancer cell lines confirmed both primer sets were human specific; however, HPRT1 was selected because it produced a single amplification product without non-specific bands. RT-qPCR is now being used to determine the limit of detection for human transcripts within RNA extracted from whole zebrafish embryos. Varying numbers of human lung cancer cells are added to zebrafish embryo RNA to determine the minimum number reliably detected using the human-specific HPRT1 primer. Establishing this detection threshold will confirm that human gene expression can be measured directly from whole embryo RNA without isolating tumor cells. This method will enable future studies examining transcriptional responses of circulating tumor cells exposed to vascular shear stress during metastasis.
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Date
1/1/2026
Student Status
Junior (Graduating in 2027)
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Poster
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Medical Laboratory Science
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College of Nursing and Health Sciences
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Life Science