The zebrafish (Danio rerio) has gained prominence as a versatile model organism in biomedical research due to several advantageous traits. These include high fecundity, transparency of early embryos, rapid embryonic development, and significant genetic similarity to humans, sharing approximately 70% gene homology and 82% conservation of disease-associated genes. These characteristics enhance the zebrafish's utility for studying various human diseases across areas such as cardiovascular health, neurological disorders, and metabolic dysfunctions.
An essential aspect of using zebrafish in research is the ability to extract genomic DNA (gDNA) efficiently for downstream applications including genotyping and sequencing. Common methods for gDNA extraction, like phenol-chloroform extraction and commercial kits, tend to offer high purity but are often time-consuming, labor-intensive, and costly. Many also involve hazardous reagents, limiting their application in high-throughput settings. Thus, a simplified and economical method is sought to facilitate routine molecular analysis.
The Chelex-100 resin has emerged as a viable alternative for gDNA extraction. This chelating resin binds divalent metal ions, inactivating nucleases and removing PCR inhibitors while disrupting cells under alkaline and boiling conditions to aid in DNA release. Historically, Chelex-based methods have been widely utilized for various organisms; however, research on its application to adult zebrafish tissues has been limited, particularly regarding obtaining DNA suitable for PCR analysis from diverse tissue types.
To bridge this gap, we developed a rapid, optimized protocol employing Chelex-100 and proteinase K for gDNA extraction from multiple adult zebrafish tissues. This method aims to support standard molecular applications efficiently.
For this study, approximately six-month-old wild-type AB zebrafish were acquired from Shandong YiXiYue Biotechnology Co., Ltd. The zebrafish were maintained under controlled conditions, following standard guidelines and housed at a constant temperature of 28 °C with a 14-hour light/10-hour dark cycle. Only healthy fish specimens were utilized in experiments, all procedures aligned with the ethical guidelines approved by the Institutional Animal Care and Use Committee of Henan University of Science and Technology.
Adult zebrafish were anesthetized with tricaine methanesulfonate before dissecting various tissues, including muscle, liver, and spleen. Samples from one male and one female were pooled into a microfuge tube for each tissue type, standardized to 3–5 mg for consistency across replicates, which were later processed for DNA extraction or stored at –80 °C.
For building the protocol, 5 mg of zebrafish muscle tissue was combined with 300 μL of 5% Chelex-100 resin and 2 μL of proteinase K. The mixture underwent a series of vortexing and centrifugation steps followed by incubation at 56 °C for various times, optimizing for 15 minutes, and boiling for 10 minutes. Each condition underwent thorough testing to ascertain the optimal extraction parameters.
The extraction involved other tissues utilizing Chelex-100 and proteinase K at defined ratios. The steps mirrored those employed for muscle tissue, with adjustments made as needed for each tissue type.
Both methods of gDNA extraction underwent evaluation using PCR, DNA sequencing, and real-time PCR (qPCR) to assess DNA quality. Primers for various target genes were utilized, and results were analyzed through gel electrophoresis.
The performance of the Chelex-100 method showed promising results, with DNA concentrations typically higher than those of commercial kits, though with lower purity levels. It provided sufficient quality for successful PCR amplification and reliable sequencing, making it suitable for applications requiring gDNA from zebrafish. Given its cost-effectiveness and operational simplicity, this protocol stands out as a strong candidate for routine applications in genotyping and molecular analysis.
The optimized Chelex-100/proteinase K protocol represents a reliable, rapid, and economical alternative for extracting gDNA from adult zebrafish tissues. Although the resulting DNA may have lower purity, its efficacy in supporting PCR and sequencing makes it particularly advantageous for many molecular research applications.