For RNA extraction, 5 mL of blood was taken from the femoral vein of cynomolgus monkeys after fasting for 14‐16 hours. White blood cells were extracted from the blood,13 and total RNA was extracted using the TRIzol method.14 , 15 , 16 Then, cDNA was synthesized from 2 μg of total RNA using the EasyScript First‐Strand cDNA Bio‐Synthesis SuperMix (TransGen Biotech) and oligo‐dT primers (Invitrogen).
Each DNA sample was amplified by PCR amplification in a 20 μL volume containing 10 μL of SYBR® Premix Ex TaqTM (2 × ), 0.4 μL of the forward primer (10 μmmol/L), 0.4 μL of the reverse primer (10 μmmol/L), 2 μL of cDNA as the template DNA, and 7.2 μL of ddH2O. The amplification and analysis were performed using an ABI StepOne™ Real‐Time PCR System and the SDS 2.3 software package (Applied Biosystems). The PCR conditions were as follows: after the initial denaturation at 95°C for 5 minutes, we performed a denaturing step at 95°C for 5 seconds and an annealing step at 60°C for 34 seconds for 40 cycles. A dissociation stage followed the system default setting at 95°C for 15 seconds, 60°C for 1 minute and then 95°C for 15 seconds.
Each DNA sample was amplified by PCR amplification in a 20 μL volume containing 10 μL of SYBR® Premix Ex TaqTM (2 × ), 0.4 μL of the forward primer (10 μmmol/L), 0.4 μL of the reverse primer (10 μmmol/L), 2 μL of cDNA as the template DNA, and 7.2 μL of ddH2O. The amplification and analysis were performed using an ABI StepOne™ Real‐Time PCR System and the SDS 2.3 software package (Applied Biosystems). The PCR conditions were as follows: after the initial denaturation at 95°C for 5 minutes, we performed a denaturing step at 95°C for 5 seconds and an annealing step at 60°C for 34 seconds for 40 cycles. A dissociation stage followed the system default setting at 95°C for 15 seconds, 60°C for 1 minute and then 95°C for 15 seconds.