Quantitative Analysis of Gene Expression in Infected Macrophages

Total RNA was extracted from infected and uninfected MDM cells at 3, 6, 12, 24, and 36 h post-infection using the RNAfast200 kit (Fastagen, Shanghai, China). cDNA was synthesized using the RevertAid First strand cDNA synthesis kit (Thermo, Waltham, MA, USA) according to the manufacturer's instructions. The primers used for quantitative real time polymerase chain reaction (qRT-PCR) have been previously reported (Kint et al., 2015 (link)). GAPDH was used as an internal control (Dai et al., 2015 (link)). qRT-PCR was performed on a Bio-Rad CFX96 Real-Time Detection System using iTaqTM Universal SYBR Green Supermix Kit reagents (BIO-RAD, California, USA) according to the manufacturer's specifications. Data analyses were performed using the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)). All experiments were performed in triplicate.