Combination of let-7d-5p, miR-26a-5p, and miR-15a-5p is suitable normalizer for studying microRNA expression in skin tissue of Liaoning cashmere goat during hair follicle cycle

The microRNAs are non-coding RNA molecules of approximately 20–22 nucleotides that are found to be implicated in a wide range of physiological processes. In this study, the suitability of 10 candidate reference RNAs was evaluated for microRNA expression data in the skin tissue of Liaoning cashmere goat including 1 small nuclear RNA (snRNA; RNU6B), 1 small nucleolar RNA (snoRNA; Z30), 1 rRNA (5S), 1 transfer RNA (tRNA; Met-tRNA), and 6 microRNAs (miR; let-7d-5p, miR-15a-5p, miR-26a-5p, miR-125a-5p, miR-214-3p, and miR-221-3p). Based on geNorm and NormFinder algorithms, we identified let-7d-5p, miR-26a-5p, and miR-15a-5p as the most stable reference RNAs. Also, three reference RNAs (let-7d-5p, miR-26a-5p, and miR-15a-5p) were sufficient for the normalization of microRNA expression data in the skin of this breed. We further assessed the suitability of let-7d-5p, miR-26a-5p, and miR-15a-5p in a combination as reference RNAs through detecting the relative expression of miR-24-3p, miR-29a-3p, miR-145a-5p, and miR-205-5p as putative genes of interest. Significant differences were revealed in the relative expression of miR-24-3p, miR-29a-3p, miR-145a-5p, and miR-205-5p at telogen stage of hair follicle cycle when a combination of let-7d-5p, miR-26a-5p, and miR-15a-5p vs a single let-7d-5p were used as reference RNA. Based on the results from this study, we suggested that the combination of let-7d-5p, miR-26a-5p, and miR-15a-5p as normalizers for microRNA expression data would be more reliable than that of single let-7d-5p, and the geometric mean of these three microRNAs (let-7d-5p, miR-26a-5p, and miR-15a-5p) can be used for the normalization of microRNAs expression data in the skin of Liaoning cashmere goat.