Quantitative RT-PCR Gene Expression Analysis

Gene expression was evaluated as previously described [46 (link)]. Total RNA was purified using the EasyPure^® RNA kit from Annagen Biotech (Baltimore, MD) or the High Pure RNA Isolation Kit from Roche Life Science (Indianapolis, IN) The cDNA prepared with TransScript^® First Strand cDNA Synthesis Supermix (Annagen Biotech) or First Strand cDNA Synthesis Kit for RT-PCR (Roche Life Science). PCR amplification was performed with an Eppendorf Realplex Instrument (Eppendorf AG, Hamburg, Germany) with SYBR Green supermix (Fermentas, Glen Burnie, MD), 0.8 μM of each primer, and 1 μl cDNA. Primer sequences are listed in Supplementary Table 1. Relative gene expression changes were calculated using the 2^−ΔΔCT method, and expression normalization was accomplished using housekeeping gene glyceraldehydes 3-phosphate dehydrogenase (GAPDH).