RNA isolation, reverse transcription, and real-time quantitative PCR
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E3 ubiquitin ligases [muscle specific F-box protein (Atrogin-1) and muscle RING-finger protein 1 (MuRF1)] were analyzed pre- (day 14) and post-immobilization (day 28) to investigate protein degradation through the ubiquitin-proteasome system (UPS) during immobilization. Total RNA was isolated from 20 mg of snap-frozen muscle tissues using the TRIzol reagent (15596–026, Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. RNA was resuspended in 33 μL of RNase-free distilled water (UltraPure water, Invitrogen, Waltham, MA, USA), and RNA concentration and purity were measured using Nano-Drop 2000 Spectrophotometer (Thermo Fisher Scientific Rockville, MD, USA). cDNA was synthesized by reverse transcription using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). 20 ng of the synthesized cDNA samples were run with triplicate 6 μL reactions containing PCR master mix (A6002, GoTaq, Promega, Madison, WI, USA). 5uM of primers (Sigma-Aldrich) of target genes [sequence 5’ to 3’; Atrogin-1, forward: 