Quantitative real-time PCR method to evaluate gene expression in zebrafish embryos

Abstract

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.