Cfx connect qpcr detection system

Muscle tissue and myoblasts were harvested, and total RNA was isolated using RNAiso reagent (TaKaRa, Dalian, China). The PrimeScript RT reagent kit (TaKaRa) was used to synthesize cDNA. Real-time quantitative PCR (RT-qPCR) was performed using the TB Green Premix Ex Taq II Kit (TaKaRa) and a CFX Connect qPCR Detection System (BIO-RAD, CA, USA). To standardize the results, GAPDH was employed as an internal reference. Relative mRNA expression was calculated using the 2^−ΔΔCt method [21 (link)].
The differentiation status of myoblasts was evaluated by detecting the mRNA levels of MYOD1 (myogenic differentiation 1), MYOG (myogenin), MYF6 (myogenic factor 6, also known as MRF4), MYH3 (myosin heavy chain 3), MYMK (myomaker, myoblast fusion factor) and CKM (creatine kinase), which are widely recognized marker genes of differentiated myoblasts and fused myotubes [22 (link),23 (link)]. To investigate the potential role of m⁶A methylases in bovine skeletal myogenesis, we selected the five most well-known m⁶A methylases, including m⁶A methyltransferases METTL3, METTL14, WTAP and m⁶A demethylases FTO and ALKBH5 and examined their mRNA expression patterns in bovine skeletal muscle and myoblasts. All primers used in RT-qPCR are listed in Table S1.

Total RNA was isolated from cells by using TRIzol reagent (Takara, Tokyo, Japan) according to the instructions of the manufacturer. Nuclear and cytoplasmic RNA fractions were separated from 10²–10⁷ cell pellets with the PARIS Kit (Ambion, Life Technologies) according to the manufacturer’s instructions. RNA sample was treated with RNase R (Geneseed) at 37°C for 30 min to obtain purified circRNA. For circRNA and mRNA, cDNA was reverse-transcribed by using HiScript II Q RT SuperMix for qPCR (R223-01, Vazyme). For miRNA, cDNA was synthesized by using PrimeScript RT Reagent Kit with gDNA Eraser (RR0471, Takara) with Bulge-Loop miRNA RT Primer (R10031.7, RiboBio). qRT-PCR was carried out using the SYBR Green Realtime PCR Master Mix (QPK-201, TOYOBO) with the CFX connect qPCR Detection System (Bio-Rad). β-actin was used as the endogenous control for mRNA and circRNAs, while U6 was used for microRNAs to calculate the relative fold changes for transcript abundance. Every experiment was carried out in three replicates. Primers sequence details are shown in Table S1.

Total RNA was isolated by phenol–chloroform extraction using peqGold RNA TriFast (PeqLab, Erlangen, Germany; Nr. 30-2020), as previously described [49 (link)]. Afterwards, purity and RNA concentration were measured using a NanoDrop 1000 device (PeqLab). For cDNA synthesis, 1 µg of RNA was used, and reverse transcription was performed using an MMLV reverse transcriptase kit and random hexanucleotide primers (Invitrogen, Braunschweig, Germany). Semiquantitative real-time PCR (qRT-PCR) was performed using SYBR Green (Bioline, Braine-Álleud, Belgium, QT615-05) in the CFX Connect qPCR detection system (Bio-Rad, Feldkirchen, Germany). The following protocol was used: 95 °C for 10 min to activate the polymerase, followed by 40 cycles of denaturation for 15 s at 95 °C, primer hybridization for 30 s (temperature as indicated in Table 1), and elongation for 30 s at 72 °C. For evaluation, the relative quantification of the measured mRNA was calculated by the ΔΔCt method using cyclophilin A as the reference gene. All primers used in this study are shown in Table 1.