RT-qPCR Analysis of Adipose Gene Expression

Total RNA was extracted from mWAT, adipocytes, or SVF using TRIsure reagent (Bioline Reagents Ltd., London, UK). cDNA was synthesized by the High Capacity cDNA Reverse Transcription Kit (Thermo Scientific) using 500 ng of total RNA. RT-qPCR was performed using StepOne Plus Real-Time PCR system with Taqman PCR master mix (Thermo Scientific). The primers used in this study were: Spred2 (Mn01223872_g1), tnfa (Mm00443258_m1), mcp-1 (Mm00441242_m1), adiponectin (Mm00456425_m1), leptin (Mm00434759_m1), pai1 (Mm00435858_m1), and rplp0 (Mm00725448_s1) (Thermo Scientific). The expression level of the gene of interest was normalized against rplp0 and expressed as fold-increases relative to the negative control for each treatment at each time point as previously described (17 (link), 33 (link)).

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Ohkura T., Yoshimura T., Fujisawa M., Ohara T., Marutani R., Usami K, & Matsukawa A. (2019). Spred2 Regulates High Fat Diet-Induced Adipose Tissue Inflammation, and Metabolic Abnormalities in Mice. Frontiers in Immunology, 10, 17.

Publication 2019

High Capacity cDNA Reverse Transcription Kit StepOne Plus Real-Time PCR system Taqman PCR master mix tnfa mcp-1 adiponectin leptin pai1 rplp0

Adipocytes Adiponectin Cdna Expression gene Leptin Mcp 1 Pai1 Primers Reverse transcription Rplp0 Spred2 Tnfa

Corresponding Organization : Okayama University

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Variable analysis

independent variables

Sample type (mWAT, adipocytes, or SVF)

dependent variables

Expression levels of Spred2, tnfa, mcp-1, adiponectin, leptin, and pai1

control variables

Rplp0 expression level (used for normalization)

controls

Negative control for each treatment at each time point

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