Nanodrop 2200

For RNA extraction from tissue, 40 to 500 mg of tissue (depending on tissue yield) were used, and total RNA was extracted with 1 mL of TRI Reagent^® Solution (Applied Biosystems, Alcobendas, Madrid, Spain). For RNA extraction from cells, the cells seeded in 3 replicate wells were pooled together at each sampling point during the culture, and total RNA was extracted with 1 mL of TRI Reagent^® Solution. The RNA concentration and purity of the samples were determined using the Nanodrop 2200TM (Thermo Scientific, Alcobendas, Madrid, Spain). The RNA integrity was checked in a 1% (w/v) agarose gel stained with SYBR-Safe^® DNA Gel Stain (Life Technologies, Alcobendas, Madrid, Spain). For cDNA synthesis, 1.1 µg of total RNA was treated with DNase I Amplification Grade (Life Technologies, Alcobendas, Barcelona, Spain) and retrotranscribed with the Transcriptor First Strand cDNA Synthesis Kit^® (Roche, Sant Cugat del Vallès, Spain) [49 (link)].

To perform the gene expression analysis, tissue homogenization was carried out from 30 or 100 mg of liver or white muscle, respectively. The samples were homogenized in 1 mL of TRI Reagent^® Solution (Applied Biosystems, Alcobendas, Spain) using Precellys^® Evolution Homogenizer cooled at 4–8 °C with Cryolys^® (Bertin Technologies, Montigny-le–Bretonneux, France). After homogenization, RNA extraction was performed following the manufacturer’s TRI Reagent^® protocol. The RNA concentration and purity of the samples were determined using the Nanodrop 2200^TM (ThermoScientific, Alcobendas, Spain). The RNA integrity was checked in a 1% (w/v) agarose gel stained with SYBR-Safe^® DNA Gel Stain (Life Technologies, Alcobendas, Spain). The RNA samples were stored at −80 °C.
For cDNA synthesis, 1.1 µg of total RNA was treated with DNase I Amplification Grade (Life Technologies, Alcobendas, Spain) and retrotranscribed with the Transcriptor First Strand cDNA Synthesis Kit^® (Roche, Sant Cugat del Vallès, Spain). The cDNA obtained was stored at −20 °C until further analysis.