Quantitative RNA Expression Analysis with qRT-PCR

RNA extraction was performed using the SV Total RNA Isolation System (Promega, Madison, WI, USA), and the RNA was subsequently digested with DNase I (Takara). The extracted RNA was reverse transcribed to cDNA using the Takara RNA PCR Kit (Takara, Kyoto, Japan). Quantitative real-time PCR (qRT-PCR) detection was performed using SYBR Premix ExTaq (Perfect Real Time; Takara Bio) on a Rotor-Gene 3000 system (Corbett Research, Sydney, NSW, Australia) to quantify gene expression levels. SAND (SGN-U316474) was employed as an internal control (Expósito-Rodríguez et al., 2008 (link)). The expression level of each gene was calculated using Rotor-Gene 6.1.81 software with two standard curves. The primer sequences are shown in Supplementary Tables S2, S3.