Laboratory of Veterinary Pharmacology and Toxicology , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing, Jiangsu Province , China .
Laboratory of Veterinary Pharmacology and Toxicology , College of Veterinary Medicine , Nanjing Agricultural University , Nanjing, Jiangsu Province , China . ; Guangdong Agribusiness Group Corporation , Guangzhou, Guangdong Province , China .
Genet Mol Biol. 2014 Sep;37(3):500-7. doi: 10.1590/s1415-47572014000400005.
Reference genes are commonly used for normalization of target gene expression during RT-qPCR analysis. However, no housekeeping genes or reference genes have been identified to be stable across different tissue types or under different experimental conditions. To identify the most suitable reference genes for RT-qPCR analysis of target gene expression in the hepatopancreas of crucian carp (Carassius auratus) under various conditions (sex, age, water temperature, and drug treatments), seven reference genes, including beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), alpha tubulin (TUBA), ribosomal protein l8 (RPL8) and glucose-6-phosphate dehydrogenase (G6PDH), were evaluated in this study. The stability and ranking of gene expression were analyzed using three different statistical programs: GeNorm, Normfinder and Bestkeeper. The expression errors associated with selection of the genes were assessed by the relative quantity of CYP4T. The results indicated that all the seven genes exhibited variability under the experimental conditions of this research, and the combination of ACTB/TUBA/EEF1A or of ACTB/EEF1A was the best candidate that raised the accuracy of quantitative analysis of gene expression. The findings highlighted the importance of validation of housekeeping genes for research on gene expression under different conditions of experiment and species.
参考基因通常用于 RT-qPCR 分析中目标基因表达的归一化。然而，尚未确定在不同组织类型或不同实验条件下稳定的管家基因或参考基因。为了确定最适合用于鲫鱼（Carassius auratus）肝胰腺中目标基因表达的 RT-qPCR 分析的参考基因，本研究评估了 7 个参考基因，包括β肌动蛋白（ACTB）、β-2 微球蛋白（B2M）、胚胎延伸因子-1α（EEF1A）、甘油醛-3-磷酸脱氢酶（GAPDH）、α微管蛋白（TUBA）、核糖体蛋白 l8（RPL8）和葡萄糖-6-磷酸脱氢酶（G6PDH）。使用三个不同的统计程序（GeNorm、Normfinder 和 Bestkeeper）分析了基因表达的稳定性和排名。通过 CYP4T 的相对数量评估了选择基因的表达误差。结果表明，在本研究的实验条件下，所有 7 个基因均表现出可变性，ACTB/TUBA/EEF1A 或 ACTB/EEF1A 的组合是提高基因表达定量分析准确性的最佳候选者。这些发现强调了在不同实验条件和物种下验证管家基因对基因表达研究的重要性。
