Tripure reagent

TriPure reagent (Sigma-Aldrich, MO) was immediately added to brain tissue homogenate collected for mRNA extraction and stored at −80°C. cDNA was synthesized using Super-Script III Reverse Transcriptase (Sigma-Aldrich, MO). Real-time PCR was performed using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Foster City, CA) and 125 nM forward and reverse primers of target genes (rat GAPDH Fwd: 5′-ACC CAG AAG ACT GTG GAT GG-3′, Rev: 5′-CAC ATT GGG GGT AGG AAC AC-3′; rat NOP Fwd: 5′-GTT CAA GGA CTG GGT GTT CAG CCA GGT AGT-3′; rat NOP Rev: 5′-TGC TGG CCG TGG TAC TGT CTC AGA ACT CTT-3′; rat preproN/OFQ Fwd: 5′-TGC ACC AGA ATG GTA ATG TG-3′, Rev: 5′-TAG CAA CAG GAT TGT GGT GA-3′, all from Sigma-Aldrich) in an ABI 7000 Sequence Detection System (Applied Biosystems, CA). GAPDH gene was used as an internal standard to which expression of other genes was normalized. Data were analyzed using the comparative Ct method and compared to control values from sham rats (Schmittgen and Livak, 2008 (link)).

RNA was isolated from mouse TA muscles and from cultured cells with TriPure reagent (Sigma‐Aldrich); 1 μg of total RNA was reverse transcribed, and 40 ng of total RNA equivalents were amplified using an iCycler iQ real‐time PCR detection system (Bio‐Rad Laboratories, Nazareth, Belgium). RT‐qPCR primers for mouse cyclophilin, interleukin 10 (IL‐10), myogenin (MyoG), myogenic regulatory factor 4 (Mrf4), myosin heavy chain 1 (Myh1) and 7 (Myh7) were used as reported.10 New mouse primer sequences were myogenic factor 5 (Myf5) (sense, 5′‐GAG GTG CAC CAC CAC CAA CCC‐3′; antisense, 3′‐CAT TCA GGC ATG CCG TCA GAG CAG‐5′) and myoblast determination protein 1 (MyoD) (sense, 5′‐CGC CGC TGC CTT CTA CGC AC‐3′; antisense, 3′‐GGG CCG CTG TAA TCC ATC ATG CC‐5′). RT‐qPCR primers for human TATA box‐binding protein, TNFα, and utrophin were similar to those previously reported.9 New human primer sequences were IL‐1β (sense, 5′‐GAA TCT CCG ACC ACC ACT ACA‐3′; antisense, 3′‐TGC ACA TAA GCC TCG TTA TCC C‐5′). The threshold cycles (Ct) were measured in separate tubes and in duplicate. The identity and purity of the amplified product were checked by electrophoresis on agarose minigels, and analysis of the melting curve was carried out at the end of the amplification. To ensure the quality of the measurements, each plate included a negative control for each set of primers.

TriPure reagent (Sigma-Aldrich, St. Louis, MO, USA) was used for hepatic total RNA isolation according to the standard procedure. The quantity and quality of isolates were monitored on a Nanodrop spectrophotometer. For reverse transcription, 2 μg of RNA was used and the reaction was performed with a Transcriptor High Fidelity cDNA Synthesis kit (Roche, Indianapolis, IN, USA). SYBR Green detection system was applied for semiquantitative mRNA transcript level measurements (LightCycler 480 SYBR Green I Master Kit, Roche, Indianapolis, IN, USA). Real-time PCR reactions were conducted on a LightCycler 480 II (Roche, Indianapolis, IN, USA). The PCR primers for the selected fragment of the Fasn gene were adopted from Sawano et al. [14 (link)]. Primer sequences for the two reference genes (Hprt, hypoxanthine-guanine phosphoribosyltransferase gene and Tbp, TATA box binding protein gene) were chosen, as described by Nowacka-Woszuk [15 (link)]. Each sample was analyzed in duplicate and quantification was performed after normalization of the Fasn mRNA level to the transcript level of the reference genes [16 ].