Zhu Xin, Li Yu-Long, Chen Dun-Xue, Wu Ping, Yi Tan, Chen Tao, Zhang Jian-She, Chu Wu-Ying
Department of Bioengineering and Environmental Science, Changsha University, Changsha 410003, China.
College of Veterinary Medicine, Hunan Agriculture University, Changsha 410128, China.
Int J Mol Sci. 2015 Apr 14;16(4):8310-23. doi: 10.3390/ijms16048310.
Real-time quantitative reverse transcription PCR (RT-qPCR) is one of the most effective and sensitive techniques in gene expression assay, for which selection of reference genes is a prerequisite. In teleost species, such as Chinese perch, the expression profiling of miRNAs as reference genes for RT-qPCR has not been intensively studied. In the present study, the expression profiles of six miRNAs (miR-101a, miR-146a, miR-22a, miR-23a, miR-26a and let-7a) and one small nuclear RNA (U6) were assayed with RT-qPCR in different adult tissues, developmental stages and growth conditions of Chinese perch, Siniperca chuatsi. The analyses revealed that embryonic developmental stage is an important variability factor in the expression stability of miRNAs. All six miRNAs exhibited better expression consistency than U6 in most of the conditions examined, and therefore, they may be more suitable as a reference gene for miRNA quantification. When different tissues and developmental stages were considered, miR-22a demonstrated the most consistent expression pattern, and the best combination of reference genes was miR-22a and miR-23a. Our study offers useful data for selecting miRNAs as reference genes for RT-qPCR analysis of miRNAs in teleost fishes under different conditions.
实时定量逆转录PCR（RT-qPCR）是基因表达分析中最有效、最灵敏的技术之一，而选择合适的内参基因是该技术的前提条件。在硬骨鱼类中，如中国鲈，尚未对作为RT-qPCR内参基因的miRNA表达谱进行深入研究。在本研究中，采用RT-qPCR技术检测了六种miRNA（miR-101a、miR-146a、miR-22a、miR-23a、miR-26a和let-7a）和一种小核RNA（U6）在中国鲈不同成体组织、发育阶段及生长条件下的表达谱。分析结果表明，胚胎发育阶段是影响miRNA表达稳定性的一个重要可变因素。在所检测的大多数条件下，六种miRNA均比U6表现出更好的表达一致性，因此，它们可能更适合作为miRNA定量分析的内参基因。综合考虑不同组织和发育阶段时，miR-22a表现出最稳定的表达模式，最佳的内参基因组合是miR-22a和miR-23a。本研究为在不同条件下选择miRNA作为硬骨鱼类miRNA的RT-qPCR分析内参基因提供了有用的数据。
