RNA Extraction and qPCR Analysis

Total RNA was extracted using TRI Reagent^® (Sigma-Aldrich). RNA (3 μg) was retro-transcripted by using M-MLV (Promega, Madison, WI). qPCR was performed in triplicate by using validated qPCR primers (BLAST), Ex TAq qPCR Premix, and the Real-Time PCR LightCycler II (Roche Diagnostics, Indianapolis, IN) as previously described [14 (link)]. mRNA levels were normalized to actin mRNA, and the relative mRNA levels were determined through the 2^−ΔΔCt method.

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Lettieri-Barbato D., Cannata S.M., Casagrande V., Ciriolo M.R, & Aquilano K. (2018). Time-controlled fasting prevents aging-like mitochondrial changes induced by persistent dietary fat overload in skeletal muscle. PLoS ONE, 13(5), e0195912.

Publication 2018

TRI Reagent M-MLV Ex TAq qPCR Premix Real-Time PCR LightCycler II

Actin Diagnostics Mrna Primers Promega Real time pcr

Corresponding Organization : University of Rome Tor Vergata

Other organizations : IRCCS Ospedale San Raffaele

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Variable analysis

independent variables

Total RNA extraction using TRI Reagent®
Reverse transcription using M-MLV

dependent variables

MRNA levels

control variables

Actin mRNA used for normalization

controls

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

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