Rotor genetm 2000

Total RNA was extracted from 3T3-L1 adipocytes using TRIzol Reagent. The cDNAs were synthesized from 4 μg RNA, using an M-MLV reverse transcriptase. Quantitative real-time PCR (qRT-PCR), was then carried out in 25 μL of Universal SYBR^® Green PCR Master Mix using a fluorometric thermal cycler (Rotor-GeneTM 2000; Corbett Research, Mortlake, NSW, Australia). Primer3 software (Version 2.3.6, Boston, MA, USA) was used for the primer design [38 ]. The sequences of the primers used are presented in Table 1. For relative quantification, the delta–delta Ct method was used [39 (link)], and β-actin was used as an endogenous control. Values presented represent fold changes compared to the control.

Total RNA was extracted from 3T3-L1 adipocytes by using TRIzol Reagent. The cDNAs were synthesized from 4 μg RNA by using M-MLV reverse transcriptase. Quantitative real-time PCR was then carried out in 25 μL of Universal SYBR^® Green PCR Master Mix using a fluorometric thermal cycler (Rotor-GeneTM 2000; Corbett Research, Mortlake, NSW, Australia). Primer3 software (Version 2.3.6, MA, USA) was used for the primer design [66 (link)]. The sequences of the primers used are presented in Table 3. For relative quantification, the delta–delta Ct method was used [67 (link)], and β-actin was used as an endogenous control. Values presented represent fold changes compared to the control.

Total RNA was isolated from epididymal WAT or skeletal muscle using Ribo Ex (Geneall Biotechnology Co., Ltd., Daejeon, Korea). The cDNA was synthesized from 4 μg total RNA using M-MLV reverse transcriptase (Bioneer Co., Daejeon, Korea). A fluorometric thermal cycler (Rotor GeneTM 2000; Corbett Research, Mortlake, N.S.W., Australia) and AccuPower 2X Greenstar qPCR Master mix (-ROX Dye) (Bioneer Co.) were utilized for Real Time qPCR analysis, and the data were relatively quantified using the ΔΔC_t method [20 (link)]. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene for normalization and the results were expressed as a fold-difference compared to the HFD group. Primers used in the present study are described in supplementary Table S1.