Validation of Housekeeping Genes for Gene Expression Profiling in Fish: A Necessity

Real-time polymerase chain reaction (qPCR) is a reliable tool for gene expression analysis in various organisms. Its use in aquaculture and study of fish physiology is increasing day by day. Relative quantification of mRNA through real-time qPCR requires suitable housekeeping genes/reference genes. Expression of housekeeping genes were thought to be constant in different tissues under various physiological and experimental conditions. In light of the literature reviewed here, it is clear that expression of most of the genes varied in different physiological conditions, under different experimental conditions, and in different gender. Use of housekeeping genes without proper validation may lead to erroneous results and misinterpretation of data. To solve this problem, various types of user-friendly software are now available for validation of housekeeping genes. Moreover, it is recommended to use more than one housekeeping gene for relative quantification. Using the geometric mean of the three most stable genes as a housekeeping gene can also solve this problem to some extent. Therefore, there is a dire need to validate a set of housekeeping genes in the species under study and in various tissues depending on the experimental design to obtain valid and reproducible results.