RNA Extraction and Real-Time PCR

Total RNA was extracted by using TRIzol (Ambion). The RNA was further purified with two phenol-chloroform treatments and then treated with RQ1 DNase (Promega) to remove DNA. The quality and quantity of the purified RNA were determined by measuring the absorbance at 260 nm/280 nm (A260/A280) using Smartspec Plus (BioRad). The integrity of RNA was further verified by 1.2% agarose gel electrophoresis. Real-time quantitative PCR (RT-qPCR) was performed for detecting gene expressions using the primers described below (Table 1). cDNA synthesis was done by standard procedures and real-time PCR was performed on the Bio-Rad S1000 (BioRad) with Bestar SYBR Green RT-PCR Master Mix (DBI Bioscience, Shanghai, China). The PCR conditions consist of denaturing at 95°C for 10 min, 40 cycles of denaturing at 95°C for 15 s, annealing, and extension at 60°C for 1 min. qPCR amplifications were performed in triplicates for each sample. Transcript levels for the genes analyzed were measured in comparison with the housekeeping gene actin as an internal reference standard, using the 2^–ΔΔCT method (14) (Livak and Schmittgen, 2001).

The genes and primers used for qRT-PCR experiments.