Quant studio tmdx

RT‒qPCR was conducted according to standard methods on SP and NSP. Briefly, total RNA was extracted from frozen muscle biopsies with Trizol reagent (Invitrogen Life Technologies, USA), and the RNA quantity was determined by NanoDrop2000 (Life Technologiess, USA). All RNA samples were homogeneous and passed quality control with 260/280 nm ratio > 1.8. Then cDNA of each sample was synthesized using 1.0 µg of RNA and the PrimeScript RT Reagent Kit (RR037A, Takara, Japan) according to the manufacturer’s instructions. The qPCR was performed by QuantStudioTM Dx real-time PCR instrument (ThermoFisher Scientific, Singapore) using TB Green Premix ExTaq II (RR820A, Takara, Japan) according to the manufacturer’s instructions. The relative gene expression was calculated using the ΔΔCt method, and GAPDH was used as the internal control. The primers used in this q-PCR reactions were listed in Additional file 1.

Furthermore, genes were determined by real-time qPCR. The gene sequences were illustrated in supplementary table 1. LO2 and HepG2 cells lines were cultured at 37°C under 5% CO₂ in Dulbecco’s modified eagle medium with 10% fetal bovine serum, and penicillin-streptomycin solution (1X). The total RNA was extracted from human liver cancer cell lines HepG2 and LO2 by the RNeasy mini kit (QIAGEN, Germany). Total RNA was further reverse transcribed to cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, China) according to the manufacturer’s instructions. We also utilized the ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, China) and applied biosystems (Quant Studio TMDx). Next, qPCR was utilized by a fluorescence qPCR instrument (ABI, Quant Studio TMDx, Thermo Fisher Scientific, Inc.). The target gene primers were applied in fluorescence quantitative qPCR analysis. The relative expression of target genes was calculated by fold change=2^-△△CT.