Selection of References for microRNA Quantification in Japanese Flounder ( Paralichthys olivaceus ) Normal Tissues and Edwardsiella tarda- Infected Livers.

MicroRNA (miRNA) plays essential roles in post-transcriptional regulation of protein coding genes, and the quantitative real-time polymerase chain reaction (qRT-PCR) is the powerful and broadly employed tool to conduct studies of miRNA expression. Identifying appropriate references to normalize quantitative data is a prerequisite to ensure the qRT-PCR accuracy. Until now, there has been no report about miRNA reference for qRT-PCR in Japanese flounder (), one important marine cultured fish along the coast of Northern Asia. In this study, combined with miRNA-Seq analysis and literature search, 10 candidates (, , , , , , , , , and ) were chosen as potential references to test their expression stability among tissues, and in livers of infected with at different time points. The expression stability of these candidates was analyzed by qRT-PCR and evaluated with Delta CT, BestKeeper, geNorm, as well as NormFinder methods, and RefFinder was employed to estimate the comprehensive ranking according to the four methods. As the result, and were proved to be the suitable combination as reference miRNAs for both normal tissues and livers infected with , and they were successfully applied to normalize and expression in livers in response to infection. All these results provide valuable information for miRNA quantitative expression analysis in the future.