Quantitative PCR Analysis of Gene Expression

Quantitative PCR was performed as previously described [6 (link)]. Briefly, total RNA was extracted by the RNeasy Plus Mini Kit (QIAGEN) and cDNA was obtained by the qScript™ cDNA SuperMix (Quanta BioSciences, Inc.). Primers were designed for these loci with the sequences freely available from the Entrez Nucleotide database and the Primer3 algorithm for primer design (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). PerfeCTa^® SYBR^® Green FastMix^® (Quanta BioSciences, Inc.) was used according to manufacturer's instructions. PCR reactions were performed on Rotor-Gene RG-3000 Real Time PCR System (Qiagen), with 18S ribosomal RNA used as endogenous control. PCR volume was 20 μl and conditions were as follow: initial cycle 50°C, 2 min, 95°C, 15 min; 45 cycles at 95°C, 20 s, 60°C, 20 s and 72°C, 20 s; final cycle 72°C, 30 s. Data were analyzed by the Rotor-Gene software using the comparative ΔΔCt method. The relative copy number was calculated based on the target gene/18S RNA ratio.