Quantifying Mature miRNA Expression

To validate the expression of mature miRNAs, miRNA expression was quantified in individual samples using a Bulge-Loop reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay (RiboBio, Guangzhou, China). One reverse transcription primer and a pair of quantitative PCR primers were designed for each of miRNAs including novel_138, oar-miR-1185-5p, novel_469, oar-miR-323c, novel_96, oar-miR-150, oar-miR-29a, and novel_459. Reverse transcription of total miRNA was conducted using 1 μg total RNA per sample and the QuantiTect Reverse Transcription Kit (Qiagen). Real-time quantitative-PCR was carried out using SYBR Green qPCR mix (Tiangen, Beijing, China), with U6 small nuclear RNA as an internal reference for normalization. Quantitative-PCR reactions were performed using a BioRad CFX96 (BioRad, CA, USA). Relative miRNA expression was calculated using the standard curve-based method for relative real-time PCR, which has been previously described [25 (link)].