Quantitative real-time polymerase chain reaction (qRT-PCR)

The expression level of Notch1 mRNA was detected by qRT-PCR. Briefly, total RNA was extracted by Trizol (Invitrogen, USA) after removing the genomic DNA using DNase I (Promega, USA) following the manufacturer’s instructions. The total RNA was reverse-transcribed to cDNA using a Reverse Transcription System (Promega, USA). Quantitative real-time PCR was performed on ABI Prism 7500 (Applied Biosystems) using SYBR® Green qPCR SuperMix-UDG (Invitrogen, USA). The cycling conditions were as follows: 2 min at 50 °C and then 2 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 30 s at 60 °C. The PCR results were quantified using the threshold cycle (Ct) method.