Quantitative Real-Time PCR Analysis

Total RNA was extracted using TRI Reagent^® (Sigma-Aldrich). Three μg of RNA was used for retro-transcription with M-MLV (Promega, Madison, WI). qPCR was performed in triplicates by using validated qPCR primers (BLAST), Ex TAq qPCR Premix, and the Real-Time PCR LightCycler II (Roche Diagnostics, Indianapolis, IN) as previously described40 (link). mRNA levels were normalized to actin mRNA, and the relative mRNA levels were determined by using the 2^−ΔΔCt method.

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Lettieri Barbato D., Tatulli G., Maria Cannata S., Bernardini S., Aquilano K, & Ciriolo M.R. (2015). Glutathione Decrement Drives Thermogenic Program In Adipose Cells. Scientific Reports, 5, 13091.

Publication 2015

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

Actin Diagnostics Mrna Primers Promega Real time pcr Transcription

Corresponding Organization :

Other organizations : University of Rome Tor Vergata, San Raffaele University of Rome

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Variable analysis

independent variables

Total RNA extraction method (using TRI Reagent®)

dependent variables

MRNA levels of target genes

control variables

Amount of RNA used for reverse transcription (3 μg)
Reverse transcription method (M-MLV)
QPCR method (triplicate, validated primers, Ex Taq qPCR Premix, Real-Time PCR LightCycler II)
Normalization to actin mRNA
Relative mRNA level quantification using 2^-ΔΔCt method

controls

No positive or negative controls were explicitly mentioned.

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