This review discusses the use of reference genes in real-time PCR (qPCR), emphasizing the importance of selecting stable genes for accurate gene expression analysis. Reference genes, often housekeeping genes, must remain consistently expressed across different conditions, tissues, and organisms. However, many genes considered stable can vary in expression under experimental conditions, leading to unreliable results. Proper selection of reference genes requires careful validation and consideration of factors such as sample preparation, primer design, and statistical analysis. The review highlights the challenges in choosing reference genes, including variability in expression, the need for validation, and the influence of experimental factors. It also discusses the use of various reference genes, such as GAPDH, 18S rRNA, and others, and their performance in different organisms and conditions. The review emphasizes the importance of validating reference genes for each study and the need for standardized protocols to ensure reliable results. It also addresses the impact of factors such as tissue type, developmental stage, abiotic stress, diseases, and tumors on gene expression, and the necessity of using appropriate reference genes for accurate analysis. The review concludes that while some genes are commonly used as references, their stability can vary, and careful validation is essential for reliable qPCR results.This review discusses the use of reference genes in real-time PCR (qPCR), emphasizing the importance of selecting stable genes for accurate gene expression analysis. Reference genes, often housekeeping genes, must remain consistently expressed across different conditions, tissues, and organisms. However, many genes considered stable can vary in expression under experimental conditions, leading to unreliable results. Proper selection of reference genes requires careful validation and consideration of factors such as sample preparation, primer design, and statistical analysis. The review highlights the challenges in choosing reference genes, including variability in expression, the need for validation, and the influence of experimental factors. It also discusses the use of various reference genes, such as GAPDH, 18S rRNA, and others, and their performance in different organisms and conditions. The review emphasizes the importance of validating reference genes for each study and the need for standardized protocols to ensure reliable results. It also addresses the impact of factors such as tissue type, developmental stage, abiotic stress, diseases, and tumors on gene expression, and the necessity of using appropriate reference genes for accurate analysis. The review concludes that while some genes are commonly used as references, their stability can vary, and careful validation is essential for reliable qPCR results.