Selection of endogenous reference microRNA genes for quantitative reverse transcription polymerase chain reaction studies of boar spermatozoa cryopreservation

It is important to select high-quality reference genes for the accurate interpretation of quantitative reverse transcription polymerase chain reaction data, in particular for certain miRNAs that may demonstrate unstable expression. Although several studies have attempted to validate reference miRNA genes in the porcine testis, spermatozoa, and other tissues, no validation studies have been carried out on cryopreserved boar spermatozoa. In this study, 15 commonly used reference miRNA genes (5S, let-7c-5p, ssc-miR-16-5p, ssc-miR-17-5p, ssc-miR-20a, ssc-miR-23a, ssc-miR-24-3p, ssc-miR-26a, ssc-miR-27a-3p, ssc-miR-92a, ssc-miR-103-3p, ssc-miR-106a, ssc-miR-107-3p, ssc-miR-186, and ssc-miR-221-3p) were selected to evaluate the expression stability of target miRNAs in boar spermatozoa under different experimental conditions and concentrations. The stability of the expression of these reference miRNAs across each sample was evaluated using geNorm, NormFinder, and BestKeeper software. The results showed that ssc-miR-186 (mean rank value = 5.00), ssc-miR-23a (5.33), and ssc-miR-27a (5.33) were the most suitable reference genes using three different statistical algorithms and comprehensive ranking. The identification of these reference miRNAs will allow for more accurate quantification of the changes in miRNA expression during cryopreservation of boar spermatozoa.