Quantitative analysis of adiponectin receptors

Real-time RT-PCR analysis was performed, as was described previously [15 (link),21 (link)]. Briefly, total RNA was extracted from muscles using the miRNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. For the detection of AdipoR1 and AdipoR2, the RNA was reverse-transcribed to cDNA using PrimeScript RT Master Mix (Takara Bio, Otsu, Japan), and then synthesized cDNA was applied to real-time RT-PCR (Thermal Cycler Dice Real Time System IIMRQ, Takara Bio) using Takara SYBR Premix Ex Taq II (Takara Bio). Relative fold change of expression was calculated by the comparative CT method with Takara Thermal Cycler Dice Real Time System Software Ver. 4.00. To normalize the amount of total RNA present in each reaction, GAPDH for AdipoR1 and AdipoR2 were used as an internal standard.
Following primers were used: adiponectin, 5’-TTCTGTCTGTACGATTGTCAGTGG-3’ (forward) and 5’-GTCATCTTCGGCATGACTGG-3’ (reverse), AdopoR1, 5’-CTGGGCATCTCTGCCATCA-3’ (forward) and 5’-CTTGACAAAGCCCTCAGCGATA-3’ (reverse); AdipoR2, 5’-ATCAGCAGCCAGACGACTC-3’ (forward) and 5’-TGACCAGTCCCAAAGACCTCTACTC-3’ (reverse); GAPDH, 5’-TGTGTCCGTCGTGTGGATCTGA-3’ (forward) and 5’-TTGCTGTTGAAGTCGCAGGAG-3’ (reverse).