Quantstudio 7 flex real time fluorescent quantitative pcr system

Total RNA was isolated using TRIzol reagent (Life Technologies, Ober-Olm, Germany) according to the manufacturer’s instructions. After quantification of the RNA concentration with Nanodrop (Thermo Scientific, Darmstadt, Germany), RNA samples were reverse transcribed at equal concentrations using a Takara First Strand cDNA Synthesis kit (Ambion, Foster City, CA) and then subjected to real-time qPCR analysis using Power SYBR Green (Applied Biosystems^® QuantStudio™ 7 Flex Real-time Fluorescent quantitative PCR system, Darmstadt, Germany). The comparative Ct method was used (2^–ΔΔCt) for quantification. The primer sequences are listed in Supplementary Table S2.

To validate the RNA-Seq data, and investigate the tissue expression pattern of the lncRNA NORHA and the expression levels of genes including caspase 3, FoxO1, miR-183, miR-96, miR-182, U6 and GAPDH, total RNA from tissues (heart, liver, spleen, lung, kidney, stomach, intestine, muscle and ovary), follicles or GCs was extracted as described, and 500 ng of total RNA was collected to synthesize first-strand cDNA of protein-coding genes using PrimeScript RT Master Mix (TaKaRa, Dalian, China). In addition, reverse transcription of miRNAs was performed with TransScript miRNA First-Strand cDNA Synthesis SuperMix (TransGen, Beijing, China). RT-PCR was performed with AceQ RT-PCRSYBR Green Master Mix (Vazyme, Nanjing, China) on a QuantStudio 7 Flex Real-Time fluorescent quantitative PCR system (Applied Biosystems, MA, USA). The expression levels of GAPDH and U6 were used as the internal controls for the protein-coding genes and miRNAs, respectively. The primer sequences used for RT-PCR are shown in Table S6.