This paper discusses the various aspects of using reference genes in quantitative real-time PCR (qPCR) techniques, which are widely used in thousands of studies. The authors emphasize the importance of selecting appropriate reference genes that meet specific criteria, such as stable expression levels and minimal variability across different tissues and physiological states. They highlight the challenges in selecting and validating reference genes, including the variability of gene expression under different conditions and the need for robust statistical analysis. The paper reviews common reference genes like GAPDH and 18S rRNA, noting their limitations and the need for validation in specific contexts. It also explores the impact of developmental stages, tissue type, environmental stress, diseases, and infections on gene expression, underscoring the importance of careful validation for each experimental setup. The authors provide a comprehensive guide to selecting and validating reference genes, emphasizing the need for a systematic approach to ensure reliable results in qPCR studies.This paper discusses the various aspects of using reference genes in quantitative real-time PCR (qPCR) techniques, which are widely used in thousands of studies. The authors emphasize the importance of selecting appropriate reference genes that meet specific criteria, such as stable expression levels and minimal variability across different tissues and physiological states. They highlight the challenges in selecting and validating reference genes, including the variability of gene expression under different conditions and the need for robust statistical analysis. The paper reviews common reference genes like GAPDH and 18S rRNA, noting their limitations and the need for validation in specific contexts. It also explores the impact of developmental stages, tissue type, environmental stress, diseases, and infections on gene expression, underscoring the importance of careful validation for each experimental setup. The authors provide a comprehensive guide to selecting and validating reference genes, emphasizing the need for a systematic approach to ensure reliable results in qPCR studies.