RT-qPCR Analysis of RNA Expression

For RT-qPCR, total RNA was extracted from the eWAT and liver by using TRIzol reagent (GeneAll Biotechnology, Seoul, Republic of Korea) [20 (link)]. cDNA was synthesized with M-MLV reverse transcriptase (Bioneer, Daejeon, Republic of Korea) by using an mRNA and cDNA synthesis kit with poly (A) polymerase tailing (ABM Inc., Richmond, BC, Canada) for miR. Next, RT-qPCR was performed with a Rotor-Gene Q thermocycler (Qiagen, Hilden, Germany) by adding Greenstar qPCR Master Mix (Bioneer). Table S2 lists the primer sequences designed by Primer3 [24 (link)], and mRNA expression was normalized using β-actin as a reference control. The specific primers of miR-34a, miR-370, and RNU6 were purchased from ABM Inc., and the expression levels of miRs were normalized using the expression of RNU6 snRNA as a control. The mRNA and miR expression levels were calculated via the 2^−∆∆Ct method for relative quantification [25 (link)]. They were then expressed as fold differences compared with those of the HF group.

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Kim J., Han D., Lee M.S., Lee J., Kim I.H, & Kim Y. (2023). Green Tea and Java Pepper Mixture Prevents Obesity by Increasing Energy Expenditure and Modulating Hepatic AMPK/MicroRNA-34a/370 Pathway in High-Fat Diet-Fed Rats. Antioxidants, 12(5), 1053.

Publication 2023

TRIzol reagent M-MLV reverse transcriptase Rotor-Gene Q thermocycler Greenstar qPCR Master Mix

Actin Cdna Liver Mrna Poly a polymerase Primers Q gene Reverse transcriptase Snrna Synthesis Trizol

Corresponding Organization : Ewha Womans University

Other organizations : Korea University

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Variable analysis

independent variables

Diet: High-fat (HF) and Control

dependent variables

MRNA expression levels of target genes
MiRNA expression levels

control variables

β-actin as a reference control for mRNA expression
RNU6 snRNA as a control for miRNA expression

controls

No specific positive or negative controls were mentioned in the input text.

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