Total RNA isolation from longissimus dorsi and biceps femoris muscle tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) was performed according to the manufacturer’s instructions. Purified and quality extracted RNA was carried out, as previously described [16 (link)].
Quantitative real-time PCR (qRT-PCR) was performed in triplicate for each complementary DNA (cDNA) sample, using an SYBR® Premix Ex Taq™ II qPCR kit (Takara Biotechnology Co. Ltd., Dalian, China) according to the manufacturer’s guidelines on an ABI7900HT real-time PCR system (Applied Biosystems, Forest City, CA, USA). Amplification was performed with the following conditions: denaturation for 10 min at 95 °C, followed by 40 PCR cycles of denaturation for 15 s at 95 °C, and annealing and extension for 60 s at 56–64 °C. Gene expression was normalized to GAPDH (internal reference) and presented as relative fold change compared with control (NP). All samples were measured in triplicate. The mRNA expression levels of target genes were calculated using the 2−ΔΔCt method [21 (link)]. All PCR primers used in this study are listed inTable 2 [22 (link),23 (link)].
Quantitative real-time PCR (qRT-PCR) was performed in triplicate for each complementary DNA (cDNA) sample, using an SYBR® Premix Ex Taq™ II qPCR kit (Takara Biotechnology Co. Ltd., Dalian, China) according to the manufacturer’s guidelines on an ABI7900HT real-time PCR system (Applied Biosystems, Forest City, CA, USA). Amplification was performed with the following conditions: denaturation for 10 min at 95 °C, followed by 40 PCR cycles of denaturation for 15 s at 95 °C, and annealing and extension for 60 s at 56–64 °C. Gene expression was normalized to GAPDH (internal reference) and presented as relative fold change compared with control (NP). All samples were measured in triplicate. The mRNA expression levels of target genes were calculated using the 2−ΔΔCt method [21 (link)]. All PCR primers used in this study are listed in
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