RNA Isolation, RT-qPCR Analysis of Gene Expression

Total RNA was isolated using RNeasy Mini Kit (QIAGEN, Hilden, Germany) from 3 million cells. Reverse transcription reaction was performed for each sample (1 μg of RNA) via iScript RT kit (Bio-Rad, Hercules, CA) per the manufacturer’s protocol. Real-time qPCR was carried out using the iTaq Universal SYBR Green PCR master mix (Bio-Rad, Hercules, CA) in a 20 μL total volume. The PCR conditions were described previously^{[19 (link)]}. The actin gene was used as an internal control to normalize the amount of amplifiable RNA. The comparative CT method was used to determine relative gene expression for each target gene. Primers were synthesized by Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO), and sequences are listed in Table 1.