Reference gene selection for microRNA expression analysis in the Pacific oyster

• Main Authors: Huang, Wen, Xu, Fei, Wang, Wei, Li, Li, Que, Huayong, Zhang, Guofan
• Format: Report
• Language: English
• Published: ARS DOCENDI 2018
• Subjects: Crassostrea gigas; MicroRNA; Real-time quantitative PCR; Reference gene; Biotechnology & Applied Microbiology; IDENTIFICATION; NORMALIZATION; BIOGENESIS; EVOLUTION; Pacific; Pacific oyster
• Online Access: http://ir.qdio.ac.cn/handle/337002/155955; https://doi.org/10.26327/RBL2018.224

Real-time quantitative polymerase chain reaction (qRT-PCR) is a widely used technique for microRNA (miRNA) expression analysis. The accuracy and reproducibility of this technique depends on the availability of stably expressed reference genes, which is absent in molluscs. Here, based on the microRNA transcriptome data of the Pacific oyster, Crassostrea gigas, candidate reference genes were selected from the stably expressed microRNAs and the commonly used housekeeping genes (U1, U6, and 5.8S). The stability of expression was evaluated and the comprehensive ranking of the reference genes was obtained using two statistical algorithms, geNorm and NormFinder. Specific combinations of reference genes were recommended for the developmental larval samples (DLS) and adult organ samples (AOS), respectively; a combination of cgi-miR-71 and Ul was recommended for DLS and a combination of cgi-let-7, cgi-miR-87, and Ul for AOS, under normal physiological conditions. The effectiveness of the reference gene combinations was further validated by quantitative analysis of two selected miRNAs in the DLS and AOS, respectively. This work was the first systematic analysis of miRNA reference gene selection in molluscs. The reference genes identified in this study will be useful for the functional analysis of miRNA in Crassostrea gigas, and also for the miRNA reference gene selection in other molluscan species.