Applied biosystems 7500 manager software v2

Total RNA was isolated from PBMCs using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 4°C for 15 min according to the manufacturer's instructions. RNA concentrations were measured using a Nano-Drop ND-1000 instrument (Thermo Fisher Scientific, Inc.). The quality of RNA was determined via the optical density 260/280 ratio. RNA integrity was measured via 1% agarose gel electrophoresis. RT was performed to synthesize the cDNA with random primers in a ReverTraAca^® RT-qPCR kit (Toyobo Life Science), which contained oligo-dT and random primers, according to the manufacturer's instructions. RT-qPCR was performed in triplicate using TB GreenTM Premix Ex Taq II (Takara Bio, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 3 min, followed by 40 cycles at 95°C for 12 sec, 62°C for 40 sec, and 72°C for 32 sec. The primer sequences are presented in Table SI. The expression level of each gene was normalized to the endogenous expression level of the β-actin transcript and was calculated using 2^−∆∆Cq method (32 (link)). The data were analyzed using Applied Biosystems 7500 Manager software v2.3 (Thermo Fisher Scientific, Inc.).