Muscle Gene Expression Analysis

A ~65 mg portion of frozen TA muscle tissue was homogenized with a Mini‐BeadBeater 8 (Biospec) while in a vial of 1 ml of TRIzol with 1.0‐mm zirconia beads (BioSpec, Cat#11079110zx). The RNAqueous phenol‐free total RNA Isolation Kit (Ambion, Cat# AM1912) was used to isolate RNA and total RNA concentration was quantified (NanoDrop 2000c, Thermo Fisher Scientific). The cDNA was synthesized utilizing 0.5 μg of RNA and the RT² First Strand Kit (Qiagen, Cat# 330401). The expression of genes relevant to growth and energy sensing was investigated using the RT² Profiler™ PCR Array for mTOR signaling (Qiagen, Cat# PARN‐098Z) per the manufacturer's instructions. Gene expression was considered significantly differentially regulated when fold regulation exceeded 1.3‐fold regulation (below 0.769‐fold change or above 1.3‐fold change) and p < 0.05(Rader et al., 2017 (link); Rader & Baker, 2020 (link)).

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Rader E.P, & Baker B.A. (2022). Elevated muscle mass accompanied by transcriptional and nuclear alterations several months following cessation of resistance‐type training in rats. Physiological Reports, 10(20), e15476.

Publication 2022

Mini‐BeadBeater 8 1.0‐mm zirconia beads RNAqueous phenol‐free total RNA Isolation Kit NanoDrop 2000c RT2 First Strand Kit

Cdna Frozen Gene expression Isolation Mtor Muscle Phenol Trizol Zirconia

Corresponding Organization : Centers for Disease Control and Prevention

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Variable analysis

independent variables

Homogenization of frozen TA muscle tissue using Mini‐BeadBeater 8 in the presence of TRIzol and zirconia beads

dependent variables

Total RNA concentration
Expression of genes relevant to growth and energy sensing

control variables

RNA extraction using the RNAqueous phenol‐free total RNA Isolation Kit
CDNA synthesis using 0.5 μg of RNA and the RT^2 First Strand Kit

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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