RNA isolation and quantitative PCR

Total RNA from gastrocnemius muscle, isolated endothelial cells, or pericytes was extracted using TRIzol (Sigma-Aldrich, T9424) according to the manufacturer’s instructions. For whole muscle, cDNA was synthesized from 1 μg of total RNA and random hexamer primers (Promega, C1181), incubated at 75°C for 7 minutes to denature RNA, and cooled at room temperature for 10 minutes. A second mixture containing M-MLV Reverse Transcriptase Buffer (ThermoFisher Scientific, 18057018), deoxynucleotide triphosphates (ThermoFisher Scientific, R0192), RNase inhibitor (ThermoFisher Scientific, 10777019), and M-MLV Reverse Transcriptase (ThermoFisher Scientific, 28025013) was added to the first mixture and incubated for 1 hour at 37°C, followed by 5 minutes at 95°C to inactivate the enzyme reaction. For isolated endothelial cells and pericytes from skeletal muscle, 300 ng and 100 ng of total RNA was reverse transcribed, respectively, using the High-Capacity RNA-to-cDNA Kit (ThermoFisher Scientific, 4387406) according to the manufacturer’s instructions. cDNA was used as a template for real-time PCR with SYBR Green Supermix (Bio-Rad, 1725121). PCR cycling conditions were 95°C for 10 minutes, 40 cycles of 95°C for 10 seconds, and 60°C for 1 minute. Quantitative determination of mRNA expression levels was performed with a LightCycler 480 Real Time PCR System (Roche) using either gene-specific primers or Rps20 gene primers for whole muscle and Gapdh gene primers for isolated cells as endogenous controls from Sigma-Aldrich (Supplemental Table 2). Samples were analyzed using the comparative CT method, where fold-change was calculated from the ΔΔCT values with the formula 2^–ΔΔCT.