Reverse transcription premix

Total mRNA was extracted from the tibialis muscle and cells using a HybridR RNA isolation kit (GeneAll, Seoul, Republic of Korea). The Reverse Transcription Premix was used to synthesize cDNA (ELPIS-Biotech, Daejeon, Republic of Korea). qRT-PCR was performed on a Light Cycler 480 Real-Time PCR System (Roche, Basel, Switzerland) using PowerUpTM SYBRTM Green Master Mix (Thermo Fisher Scientific) with gene-specific primers (summarized in Table 1). To account for potential variations, target mRNA expression was normalized against the housekeeping gene mouse β-actin. Relative gene expression was then calculated using the 2^−ΔΔCt method as previously described [22 (link)].

Total RNA was isolated from cells using TRIzol reagent (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. cDNA was synthesized from 1 μg total RNA using the Reverse Transcription Premix (Elpis-Biotech, Daejeon, South Korea) under the following reaction conditions: 45 °C for 45 min and 95 °C for 5 min. Gene expression was quantified using qPCR, and data were analyzed using the StepOne Plus™ software (Applied Biosystems, Foster City, CA, USA). qPCR amplification reactions were performed using SYBR Green PCR Master Mix with premixed ROX (Applied Biosystems, Foster City, CA, USA). The following primer pairs (Bioneer, Daejeon, South Korea) were used in the ABI 7300 Cycler internal reaction according to the manufacturer’s protocol (Online Resource 7). The reaction conditions were as follows: 40 cycles for 2 min at 50 °C, 10 min at 95 °C, 10 s at 95 °C, and 1 min at 60 °C. 18S rRNA was used as an internal control.

Total RNA was isolated using TRIzol reagent (TaKaRa; Shiga, Japan), and approximately 2 µg of total RNA was synthesized to cDNA using Reverse Transcription Premix (Elpis-biotech; Daejeon, Korea Gene expression signals were quantified, and the data were analyzed using the StepOne Plus^TM system software (v2.3, Applied Biosystems; Foster City, CA, USA). RT-qPCR amplification reactions were performed in a SYBR Green PCR Master Mix with premixed ROX (Applied Biosystems; Foster City, CA, USA). The following primer pairs (Bioneer, Daejeon, Korea) were used in the reactions performed in an ABI 7300 following the manufacturer’s protocol: β-actin (F: 5′-GGCCATCTCTTGCTCGAAGT-3′ and R: 5′-GACACCTTCAACACCCCAGC-3′), adiponectin (F: 5′-AAGGACAAGGCCGTTCTCT-3′ and R: 5′-TATGGGTAGTTGCAGTCAGTTGG -3′), PPARγ (F: 5′-AAACTCTGGGAGATTCTCCT-3′ and R: 5′-TGGCATCTCTGTGTCAAC-3′), SREBP-1a (F: 5′-GCTGCTGACCGACATCGAA-3′ and R: 5′-TCAAATAGGCCAGGGAAGTCA-3′), C/EBPα (F: 5′-GCCAAACTGAGACTCTTC-3′ and R: 5′-TGGCATCTCTGTGTCAAC-3′), and MMP-1 (F: 5′-CGAATTTGCCGACAGAGATGA-3′ and R: 5′-GAATTTGCCGACAGAGATGA-3′). The mRNA expression of β-actin was used as an internal control.