Kitap Bölümü KoleksiyonuBook Chapter Collectionhttps://hdl.handle.net/20.500.12511/41712024-03-28T12:24:44Z2024-03-28T12:24:44ZQuantitative real-time PCR method to evaluate gene expression in zebrafish embryosBeler, MerihCansız, DeryaÜnal, İsmailEmekli Alturfan, Ebruhttps://hdl.handle.net/20.500.12511/122952024-02-21T08:43:46Z2024-01-01T00:00:00ZQuantitative real-time PCR method to evaluate gene expression in zebrafish embryos
Beler, Merih; Cansız, Derya; Ünal, İsmail; Emekli Alturfan, Ebru
The fast zebrafish embryonic development offers an opportunity for the study of genes crucial for developmental processes. Several genes associated with human diseases have orthologs in zebrafish. Enhanced comprehension of a gene’s function can be achieved by knowing when and where it is expressed. This knowledge also makes it possible to alter a gene-by-gene knockdown in a time- and place-specific manner. Moreover, gene expression analyses contribute greatly to teratogenicity studies in zebrafish embryos. Therefore, the importance of examining the differences between the expressions of these genes has increased day by day. The incorporation of reverse transcription (RT) as the initial step prior to thermal cycling in quantitative real-time polymerase chain reaction (RT-qPCR) has made a significant contribution to RNA research. RT-qPCR is the gold standard and an effective method for gene expression analysis. Quick readout, high sensitivity, reproducibility, as well as high potential throughput, along with reliable quantification, are just a few advantages of RT-qPCR. However, there are drawbacks to its application, such as RNA’s inherent variability, impurities during RNA extraction, and variations in reverse transcription and PCR efficiencies. Implementing a precise normalization technique is crucial to account for these inaccuracies. In this chapter, the protocol for gene expression analysis by RT-qPCR for zebrafish embryos is explained.
2024-01-01T00:00:00ZAlcian blue staining for chondrocranium development in zebrafishBeler, MerihÜnal, İsmailCansız, DeryaEmekli Alturfan, Ebruhttps://hdl.handle.net/20.500.12511/122942024-02-21T07:57:05Z2024-01-01T00:00:00ZAlcian blue staining for chondrocranium development in zebrafish
Beler, Merih; Ünal, İsmail; Cansız, Derya; Emekli Alturfan, Ebru
Craniofacial abnormalities are one of the most frequent birth malformations in humans, affecting around one in every thousand live births. The zebrafish (Danio rerio), a model organism that has seen increased usage in toxicological research in recent years, is ideal for assessing the effects of various chemicals on bone and cartilage structures. Chondrogenesis developed in zebrafish embryos by embryonic day 2, and supporting cartilage components are apparent at hatching (72 h post-fertilization). Individual cartilage may be observed using Alcian Blue staining as early as 2 days post-fertilization (dpf). The preferential binding of Alcian Blue causes the staining of zebrafish cartilage to acidic glycoproteins in an acidic solution (pH 2.2). In 72–120 hpf embryos, the cranial skeleton is easily visible after cartilage staining using Alcian Blue. Various cranial lengths and structures can be determined by measuring specific distances and angles to optimize the quantitative analysis of cranial malformations in zebrafish after exposure to various toxic agents. This chapter explains the Alcian Blue staining procedure to identify craniofacial cartilaginous structures in zebrafish embryos.
2024-01-01T00:00:00ZWhole-mount RNA in situ hybridization of zebrafish embryosÜnal, İsmailCansız, DeryaBeler, MerihAlturfan, Ahmet AtaEmekli Alturfan, Ebruhttps://hdl.handle.net/20.500.12511/122932024-02-20T13:22:04Z2024-01-01T00:00:00ZWhole-mount RNA in situ hybridization of zebrafish embryos
Ünal, İsmail; Cansız, Derya; Beler, Merih; Alturfan, Ahmet Ata; Emekli Alturfan, Ebru
A commonly employed technique in molecular biology to evaluate the temporal and spatial expression of a certain gene is in situ hybridization. This method is an effective strategy to construct synexpression groups, co-expressed genes acting in shared biological processes, and to find new members of genes engaged in the same signaling pathways to discover similar spatial and temporal expression patterns in zebrafish embryos. The major disadvantage of this method is that RNA probes can penetrate within 2 days of post-fertilization embryos, and therefore, in later developmental stages, the probe can only reach the surface tissues. Further application of the method in histological sections will be required for a complete and accurate gene expression investigation. However, this method is highly effective at late embryogenesis and early larval stages for observing gene expression in endodermal derivatives and sensory organs. RNA probes for in situ hybridization can be prepared through in vitro transcription from plasmids carrying specific promoter elements and mRNA-specific cDNA, or an alternative polymerase chain reaction (PCR) method can be used through PCR amplification. This chapter describes the procedures for detecting gene expression in zebrafish embryos using whole-mount RNA in situ hybridization.
2024-01-01T00:00:00ZMicroarray analysis to determine gene expression changes in zebrafish embryosÜnal, İsmailBeler, MerihCansız, DeryaEmekli Alturfan, Ebruhttps://hdl.handle.net/20.500.12511/122902024-02-20T08:36:00Z2024-01-01T00:00:00ZMicroarray analysis to determine gene expression changes in zebrafish embryos
Ünal, İsmail; Beler, Merih; Cansız, Derya; Emekli Alturfan, Ebru
Chemical exposure in humans begins from the zygote stage and continues throughout the development of the embryo and the fetus. Zebrafish are one of the most powerful model organisms used in many research areas, including genetics, environmental toxicology, development, DNA damage and repair, cancer, and other diseases. Among the advantages that facilitate the use of zebrafish as a model for studies are features such as high homology with the human genome, small size, and high reproductive potential in short periods. The use of zebrafish embryos in research has increased rapidly due to their advantageous properties, including extrauterine development and the transparent feature of the embryos. However, there are thousands of genes that can be encountered in research, and in this case, the workforce is too much. This workload has been alleviated with the developed technologies. Microarray is one of these technologies. An important parameter in this assay is the RIN value. The RIN value ranges from 1 to 10, indicating mRNA degradation, and therefore helps to decide whether to continue the study. In this chapter, microarray analysis, which is one of the main techniques used in the determination of gene expression in zebrafish embryos, is described.
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