Prostate Cancer Gene Expression Analysis by qRT-PCR

Total RNA was extracted using TRIZOL and used to generate cDNA for gene expression experiments [52 (link)]. Expression of target gene mRNA transcripts was determined by Realtime PCR with gene-specific primers and SYBR-green PCR mix (Life Technologies). The primer sequences were: forward 5′-AGCCCACGCTCAGTGTCTAT-3′ and reverse 5′-GGGCACTAGGATCATCTGTCA-3′ for RON; forward: 5′-AAGCTGACTTCTTCTGGAGCCTGT-3 and reverse 5′-TCTCCTTGGCAGAAACTCTGCTGT-3 for c-FLIP′; forward: 5′-GGCACCCAGCACAATGAAGATCAA-3′ and reverse: 5′-AGAAGCATTTGCGGTGGACGATG-3′ for β-actin; forward: 5′-ACACTGCCAACTGGCTGGAGATTA-3′; reverse: 5′ TGATTAGGGCTGTGTACGTGCTGT-3 for E-cadherin′; forward: 5′-TAACCCAAGGAGCAGGTAATCGCA-3 and reverse: 5′-GTTTCTTGCAGTTTGGGCACTCGT-3′ for ZEB-2; forward 5′-GATTGAGCATGGCTCTCTATTC-3 and reverse 5′ GGTGAGATGTTCCAGGTTTAAG-3 for FKBP1; forward 5′-CATGTGATGTCTGGTCTGAAT-3 and reverse 5′-GACACAGCTCAACAAAGAAAC-3 for PMEPA1. The relative expression changes of individual genes were determined using comparative c_t method. Data is expressed as gene expression changes relative to β-actin control. Changes in expression of RON and c-FLIP were analyzed using Origene PCA II cDNA array HPRT 302 and 303 (for RON and c-FLIP expression respectively) containing different Gleason grade human prostate tumors.