RNA Extraction and RT-qPCR Analysis

RNA was extracted from fresh or frozen tissues by homogenization in TRIzol reagent (Invitrogen) as previously described (Zhu et al., 2016 (link)). We used 1 μg RNA to transcribe cDNA with a reverse transcription kit (Bio-Rad). Most of RT-qPCR primers were from the Harvard Primer Bank (https://pga.mgh.harvard.edu/primerbank/). The relative expression levels were calculated using the comparative threshold cycle method, normalized to the housekeeping gene Gapdh.

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Li N., Zhao S., Zhang Z., Zhu Y., Gliniak C.M., Vishvanath L., An Y.A., Wang M.Y., Deng Y., Zhu Q., Shan B., Sherwood A., Onodera T., Oz O.K., Gordillo R., Gupta R.K., Liu M., Horvath T.L., Dixit V.D, & Scherer P.E. (2021). Adiponectin preserves metabolic fitness during aging. eLife, 10, e65108.

Publication 2021

TRIzol reagent reverse transcription kit

Cdna Frozen Gapdh Housekeeping gene Primer Reverse transcription Tissues Trizol

Corresponding Organization :

Other organizations : Touchstone (China), Tianjin Medical University General Hospital, The University of Texas Southwestern Medical Center, Yale University

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Variable analysis

independent variables

Fresh or frozen tissues

dependent variables

Relative expression levels of genes

control variables

Homogenization in TRIzol reagent
Reverse transcription to cDNA
Housekeeping gene Gapdh for normalization

controls

No positive or negative controls were explicitly mentioned.

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