Selection of the Most Stable Endogenous Control Genes for Microrna Quantitation in Chicken Ovarian Follicles

MicroRNAs (miRNAs or miRs) belong to a class of small non-coding RNAs of 19 to 24 nucleotides long that act as negative gene regulators at the post-transcriptional level. Quantitative PCR (q-PCR) is a commonly used technique in the profiling of miRs, and identification of reliable endogenous controls is crucial for proper data normalisation. To date, no study has been performed on reference miRs for the normalisation of miR expression in chicken ovarian tissues. Therefore, the aim of the present study was to experimentally identify the most stable reference mirs for normalisation of miR q-PCR expression data in the chicken ovary. Relying on high-throughput sequencing, five putative reference miR (let-7a-3p, miR-140a-3p, miR-22-5p, miR-33-5p, miR-99a-3p) were identified and subsequently analysed in a total of 66 tissue samples. The stability of candidate endogenous controls validated by the most widely used algorithms, geNorm, NormFinder, and BestKeeper, showed that let-7a-3p, miR-140a-3p, and miR-22-5p are the most appropriate choice of reference genes. Application of different normalisation approaches to the relative quantitation of randomly chosen miR-1552-5p in chicken ovarian follicles indicated the impact of the selected reference genes on miR expression. Further, the results revealed a downregulation of miR-1552-5p. In summary, the three identified endogenous reference miRs are suitable for profiling the miR expression in ovarian tissues of laying hens. Our findings provide valuable information for future miR expression studies in the avian ovary.