RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted from cultured cells using TRIzol (Invitrogen) according to the instructions provided by the manufacturer. We employed a Prime Script RT Reagent Kit (TAKARA, Dalian, China) to perform the reverse transcription. The target gene was amplified in a final volume of 20 μL with a SYBR Green PCR Master mix (TAKARA). The qRT-PCR reaction was run in an Applied Biosystems 7500 Real-Time PCR System (Waltham, MA), and data were collected automatically. Forward and reverse primers for all genes are given in Supplementary Table 2. The expression of target genes was normalized to that of β-actin, and relative absolute amounts of target genes were calculated according to our previous method for statistical analysis (24).