Rneasy fibrosis mini kit

RNA extraction was performed as described previously. Tissue was dissociated using Precellys® 24 tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) and total RNA purified using the automated QIAcube with RNeasy Fibrosis Mini kit (Qiagen, CA, United States) according to the manufacturer’s instructions. The RNA integrity was assessed with the Bioanalyzer 2,100 system (Agilent, CA, United States), and RIN values > 8 accepted as suitable for PCR profiling. RNA concentrations were then measured using the Nanodrop 2000 (Labtech International, East Sussex, United Kingdom).

Total RNA was extracted from soleus muscle homogenate using an RNeasy Fibrosis Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The purity and quantity of total RNA were assessed using a NanoDrop system (NanoDrop Technologies, Wilmington, DE). Total RNA was reverse transcribed into cDNA using a high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA) according to the manufacturer’s instructions. PCR was performed with a 7300 real-time PCR system (Applied Biosystems) using PowerUp SYBR Green PCR Master Mix (Applied Biosystems). The thermal profiles consisted of denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and annealing at 60°C for 60 s. The expression of β-actin mRNA was used as a housekeeping control, and all data were normalized to the β-actin mRNA level. Data are expressed as fold change relative to the values of ND/Sed group. Specific PCR primer pairs for each gene are shown in Table 2.