Adipose Tissue RNA Extraction and Gene Expression Analysis

Total RNA from human adipose tissues, mouse adipose tissues and cells were extracted using Trizol reagent (TAKARA). cDNA was synthesized using a PrimeScript RT Reagent Kit (TAKARA). Quantitative Real-Time PCR was performed on a Roche LightCycler 480 Real-Time PCR system. Mouse Arbp was used as internal control in cells and mouse adipose tissues, and human 18S was chosen as normalization control in human adipose tissues. Protein expressions were detected by western blot as previously reported (Cai et al. 2019 (link)). The primary antibodies were specific for Sterol Regulator Elementing Binding Protein 1c (SREBP1C) (Abclonal), adiponectin (CST), peroxisome proliferator-activated receptor γ (PPARγ) (CST), CCAAT-enhancer-binding protein α (C/EBPα) (Abcam), uncoupling protein 1 (UCP1) (Santa), PPARγ coactivator-1α (PGC1α) (Santa), Wnt Family Member 10B (Wnt10B) (Abclonal), SMAD Family Member 2 (SMAD2) (CST), phosphor-SMAD2 (CST), and β-ACTIN server as loading control.

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Wu C., Fang S., Zhang H., Li X., Du Y., Zhang Y., Lin X., Wang L., Ma X., Xue Y, & Guan M. (2022). Long noncoding RNA XIST regulates brown preadipocytes differentiation and combats high-fat diet induced obesity by targeting C/EBPα. Molecular Medicine, 28, 6.

Publication 2022

Trizol reagent PrimeScript RT Reagent Kit LightCycler 480 Real-Time PCR system SREBP1C PPARγ

Actin Adiponectin Adipose tissues Antibodies Binding protein Ccaat enhancer binding protein α Cdna Cells Family member Human Mouse Peroxisome proliferator activated receptor Pgc1α Phosphor Protein Quantitative real time pcr Smad2 Sterol Trizol Uncoupling protein 1 Western blot Wnt10b

Corresponding Organization :

Other organizations : Southern Medical University, Nanfang Hospital

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

MRNA expression levels of Arbp, 18S, SREBP1C, adiponectin, PPARγ, C/EBPα, UCP1, PGC1α, Wnt10B, SMAD2, and phosphor-SMAD2
Protein expression levels of SREBP1C, adiponectin, PPARγ, C/EBPα, UCP1, PGC1α, Wnt10B, SMAD2, and phosphor-SMAD2

control variables

Arbp as internal control for mouse adipose tissues and cells
18S as normalization control for human adipose tissues
β-ACTIN as loading control for western blot

positive controls

None specified

negative controls

None specified

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