Stepone plus system software

Total RNA was isolated from cell pellets using Trizol reagent and quantified by spectrophotometry. The cDNA was synthesized in a 20 µL reaction containing 2 µg of total RNA, oligo (dT), and Reverse Transcription Premix under the following reaction conditions: 45 °C for 45 min and 95 °C for 5 min. Gene expression signals were quantified using real-time RT-PCR. The data were analyzed using the StepOne Plus^TM system software (Applied Biosystems, Foster City, CA, USA). RT-qPCR amplifications were performed using SYBR Green PCR Master Mix with premixed ROX (Applied Biosystems, Foster City, CA, USA) and primers (Bioneer, Daejeon, Korea) in an ABI 7300 following the manufacturer’s protocol. Reaction conditions were as follows: initiation at 95 °C for 10 min, followed by cycling conditions of 95 °C for 15 sec, 55 °C for 30 sec, and 72 °C for 30 sec for 40 cycles. The expression of β-actin was used as an internal control.

To isolate and quantify the total RNA from the cell pellets, Trizol reagent was used, and the analysis was done using a spectrophotometer. The synthesis of cDNA was carried out in a total reaction volume of 20 µL; the reaction mixture consisted of 2 µg of total RNA, oligo (dT), and reverse transcription premix under the following reaction conditions: 45 °C for 45 min, followed by 95 °C for 5 min. RT-PCR was used for quantification of gene expression, and the results were subsequently analyzed using the StepOne PlusTM system software (Applied Biosystems, Foster City, CA, USA). RT-PCR amplifications were conducted using SYBR Green PCR Master Mix with premixed ROX (Applied Biosystems, Foster City, CA, USA) and primers (Bioneer, Daejeon, Korea) in an ABI 7300 instrument following the manufacturer’s protocol. The reaction conditions were as follows: initiation at 95 °C for 10 min, followed by cycling conditions of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s for 40 cycles. β-actin 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.