Goat Gene Expression Analysis via RT-qPCR

Total RNA from six samples (three goats) was extracted with TRIzol (Invitrogen, CA) according to the manufacturer’s instructions. Total RNA was reverse-transcribed to cDNA using the PrimeScript RT reagent Kit (TaKaRa, Tokyo, Japan) and RT-qPCR was performed using SYBR Premix Ex Taq (TaKaRa, Tokyo, Japan). The mRNA primers were synthesized by Shanghai Shenggong, China (Supplementary Table 9). The relative mRNA levels were normalized to β-actin (Hou et al., 2016 (link)) expression for each sample; For miRNA detection, reverse transcription followed by stem-loop RT-qPCR was performed according to the manufacturer’s protocols using the Bulge-LoopTM miRNA RT-qPCR Primer (RiboBio, Guangzhou, China). Quantification of miRNA was performed with a stem-loop real-time PCR miRNA kit (RiboBio). The relative miRNA levels were normalized to U6 small nuclear RNA (Carvalho et al., 2019 (link)) expression for each sample.