Quantitative real-time PCR
Total RNA was extracted from genetically modified or cultured cells using TRIzol reagent (Invitrogen). cDNA was synthesized using an RT kit (QIAGEN, USA). Relative levels of NKG2D and transcription factors were examined using an SYBR green real-time quantitative reverse transcription-PCR kit (QIAGEN) and normalized to β-actin RNA. The primer sequences of the target genes are listed in Table 1. PCR was performed at 95°C for 4 min, followed by 35 cycles of 94°C for 20 sec, 60°C for 30 sec, and 70°C for 30 sec. All procedures were performed according to the instructions provided by the manufacturer. The fold-change of each RNA was calculated using the 2ΔΔCt method.
Table 1. Primer sequences