C2C12 myoblasts were collected using TRIzol for RNA extraction. The muscles were harvested and homogenized for RNA extraction using mortar and TRIzol (Invitrogen, 15596026). Then, 1 μg of RNA was subjected to reverse transcription to cDNA using the PrimeScript Real-Time Master Mix (TaKaRa, Japan). Power SYBR Green PCR Master Mix (Thermo Scientific) was applied for the qPCR of the target mRNA detection. The relative expression level of the gene of interest normalized to β-actin was calculated in accordance with the 2-ΔΔCT formula. The sequences of the primers used for real-time PCR assay are listed in table S1. The cycling conditions were denaturation at 95°C for 10 min, 40 cycles at 95°C for 15 s, optimal annealing temperature for 20 s, and 72°C for 30 s.
Primescript real time master mix
Manufactured by Takara Bio
Sourced in Japan, China
PrimeScript Real-Time Master Mix is a reagent used for real-time quantitative PCR (RT-qPCR) experiments. It contains all the necessary components for efficient and sensitive RNA detection and quantification.
Automatically generated - may contain errors
2 protocols using primescript real time master mix
1
Quantitative Analysis of Myoblast Gene Expression
2
Quantitative Analysis of miRNA and mRNA
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Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). For the detection of miRNAs, 1000 ng RNA was subjected to reverse transcription (RT) using an miRNA RT kit (TaKaRa, Dalian, China) and used according to the manufacturer's instructions. Real-time PCR was performed using the cDNA as a template with the SYBR PrimeScript miRNA Real-Time PCR Kit (TaKaRa, Dalian, China) according the manufacturer's instructions with following primers: miR-24-3p 5'-CCCATTCAGCAGGAACAGAAA-3'; U6 5'-ACGCAAATTCGTGAAGCGTT-3'. For the detection of mRNA, cDNA was synthesized from 1000 ng RNA using the PrimeScript RT reagent kit (Takara, Dalian, China) according to the manufacturer's instructions. Real-time PCR was performed using the cDNA as a template with the PrimeScript Real Time Master Mix (TaKaRa, Dalian, China) on a real-time PCR machine (Bio-Rad CFX 96, Hercules, CA, USA) with the following primers: PDGFRB 5'-GGCTACATGGACATGAGCAAGG-3' (forward), 5'-AGCTTAGCACTGGAGACTCGTTGA-3' (reverse); C-myc 5'-GCAGCTGCTTAGACGCTGGA-3' (forward), 5'-CGCAGTAGAAATACGGCTGCAC-3' (reverse); GAPDH 5'-GCACCGTCAAGGCTGAGAAC-3' (forward), 5'-TGGTGAAGACGCCAGTGGA-3' (reverse). Data analysis was performed by the comparative C T method using Bio-Rad Manager 2.1 software (Hercules, CA, USA).
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