Thermal cycler dice real time system iimrq

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).

Real-time RT-PCR analyses were performed as was described previously [35 (link)]. Briefly, total RNA was extracted from muscles using the miRNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. For the detection of mRNA, 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. To normalize the amount of total RNA present in each reaction, S18 ribosomal RNA (18S rRNA) was used as an internal standard. The following primers were used: PGC1α (Ppargc1a), 5′-GCTGCATGGTTCTGAGTGCTAAG-3′ (forward) and 5′-AGCCGTGACCACTGACAACGAG-3′ (reverse); 18S rRNA, 3′-ACTCAACACGGGAAACCTCA-5′ (forward) and 3′-AACCAGACAAATCGCTCCAC-5′ (reverse).

Primers were designed using the Takara Bio Perfect Real Time Support System (Takara Bio, Table 2). Primers were diluted to 50 µM in ddH₂O and stored at −20 °C. Real-time RT-qPCR was performed on the cDNA (Thermal Cycler Dice Real Time System IIMRQ, Takara Bio) using Takara SYBR Premix Ex Taq II (Takara Bio. Reference: RR802A). 12.5 µL of SYBR Premix Ex were added to each RT-qPCR well. 8.5 µL of ddH₂O and 2 µL of the corresponding primers were then added (a final concentration of 2 µM per primer). 2 µL of the respective cDNA was then added to the appropriate wells, bringing the total volume to 25 µL per well. The RT-qPCR cycle consisted of 95 °C for 30 seconds (s) (for enzyme activation), followed by 40 cycles at 95 °C for 5 s and a qPCR amplification period of 30 s at 60 °C. The relative fold change of expression was calculated by the comparative threshold cycle (CT) method using Takara Thermal Cycler Dice Real Time System Software Ver. 4.00 (Takara Bio). To normalise for the amount of total RNA present in each reaction, Gapdh was used as an internal standard.