Determination of reference microRNAs for relative quantification in grass carp (Ctenopharyngodon idella)

• miRNA expression levels were quantified by quantitative real-time RT-PCR.
• Stabilities of reference gene miRNA expression levels were evaluated.
• miR-101a was the most stable miRNA across all tissues and developmental stages.
• miRNA stability values generally improved when tissues were examined separately.

Relative quantification is the strategy of choice for processing real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) data in microRNA (miRNA) expression studies. Normalization of relative quantification data is performed by comparison to reference genes. In teleost species, such as grass carp (Ctenopharyngodon idella), the determination of reference miRNAs and the optimal numbers of these that should be used has not been widely studied. In the present study, the stability of seven miRNAs (miR-126-3p, miR-101a, miR-451, miR-22a, miR-146, miR-142a-5p and miR-192) was investigated by RT-qPCR in different tissues and in different development stages of grass carp. Stability values were calculated with geNorm, NormFinder, BestKeeper and Delta CT algorithms. The results showed that tissue type is an important variability factor for miRNA expression stability. All seven miRNAs had good stability values and, therefore, could be used as reference miRNAs. When all tissues and developmental stages were considered, miR-101a was the most stable miRNA. When each tissue type was considered separately, the most stable miRNAs were 126-3p in blood and liver, 101a in the gills, 192 in the kidney, 451 in the intestine and 22a in the brain, head kidney, spleen, heart, muscle, skin and fin. 126-3p was the most stable reference miRNA gene during developmental stages 1–5, while 22a was the most stable during developmental stages 6–18. Overall, this study provides valuable information about the reference miRNAs that can be used to perform appropriate normalizations when undertaking relative quantification in RT-qPCR studies of grass carp.