2.4. Cell Culture and RNA Isolation and Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)

The C2C12 cells were procured from Procell Life Science & Technology Co., Ltd. The 293T cells were procured from Procell Life Science & Technology Co., Ltd. (Procell Life Science & Technology Co., Ltd., Beijing, China). These C2C12 cells, the bovine muscle cells, and the 293T cells were maintained at 37 °C in a 5% CO₂ humidified incubator and were grown in Dulbecco’s Modified Eagle’s Medium (DEME) supplemented with 10% fetal bovine serum (FBS) [29]. When the confluence of cells reached 80–90%, they were digested with 0.25% trypsin and passaged to a new culture dish at a ratio of 1:3. The cells were transfected with 50 nM of control or mimic for miR-24b-3p mixed with Opti-MEM and Lipofectamine RNAiMAX (Invitrogen, Bao Bioengineering (Dalian) Co., Ltd., Dalian, China) according to manufacturer’s protocol [30]. All the analyses were performed in triplicate. The cells were lysed, and total RNA was extracted using TRIzol reagent (Takara, Bao Bioengineering (Dalian) Co., Ltd., Dalian, China) according to the manufacturer’s protocol and transcribed into cDNA using a Reverse Transcription Kit (Takara, Bao Bioengineering (Dalian) Co., Ltd., Dalian, China) [31]. The expression patterns of the target genes and the transcriptional responses of the target genes to the muscle were investigated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). According to the manufacturer’s instructions, after DNase treatment, 1000 ng of total RNA was reverse-transcribed to single-strand cDNA using a HiScript^® III 1st Strand cDNA Synthesis Kit (+gDNA wiper)(Nanjing novozan Biotechnology Co., Ltd., Nanjing, China). The primer pairs of the target genes were used (Tables S2–S5). Before RT-qPCR analysis, the standard curves for the primer pair of the target genes were generated by regression of the Cq values and a series of ten-fold cDNA dilutions. Primer amplification efficiency was calculated from the slope of the corresponding standard curve, and the efficiency of the target genes. The hypoxic-stable reference gene β-actin was used as the control (Tables S2–S5). RT-qPCR was performed using the ChamQTM Universal SYBR^® qPCR Master Mix (Nanjing novozan Biotechnology Co., Ltd., Nanjing, China) with the following thermal cycling conditions: 95 °C for 30 s, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. Each experiment was performed independently three times. The relative expression levels of the target genes were normalized to that of β-actin quantification using the 2^−△△Ct method [31].