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.

Clock genes are considered to be the molecular core of biological clock in vertebrates and they are directly involved in the regulation of daily rhythms in vertebrate tissues such as skeletal muscles. Fish myotomes are composed of anatomically segregated fast and slow muscle fibers that possess different metabolic and contractile properties. To date, there is no report on the characterization of the circadian clock system components of slow muscles in fish. In the present study, the molecular clock components (clock, arntl1/2, cry1/2/3, cry-dash, npas2, nr1d1/2, per1/2/3, rorα and tim genes) and their daily transcription levels were characterized in slow and fast muscles of Chinese perch (Siniperca chuatsi). Among the 15 clock genes, nrld2 and per3 had no daily rhythmicity in slow muscles, and cry2/3 and tim displayed no daily rhythmicity in fast muscles of the adult fish. In the slow muscles, the highest expression of the most clock paralogs occurred at the dark period except arntl1, nr1d1, nr1d2 and tim. With the exception of nr1d2 and tim, the other clock genes had an acrophase at the light period in fast muscles. The circadian expression of the myogenic regulatory factors (mrf4 and myf5), mstn and pnca showed either a positive or a negative correlation with the transcription pattern of the clock genes in both types of muscles. It was the first report to unravel the molecular clock components of the slow and fast muscles in vertebrates. The expressional pattern differences of the clock genes between the two types of muscle fibers suggest that the clock system may play key roles on muscle type-specific tissue maintenance and function.