Biosystems 7900ht fast real time pcr system

RNA was extracted in accordance with manufacturer’s protocol (miRNeasy; Qiagen), and RNA-sequencing was performed as previously described (25 (link)). In brief, RNA illumine sequencing reads were de-multiplex, aligned against human genome (hg19), and results aligned to BAM formatted sequence alignment map via cufflinks program. Differential expressed transcripts were identified between 786-0 and 786-0R samples, and ranked based on the square root of the sum of squares for the log2 fold change. For qRT-PCR analysis, gene expression assessment on AR target genes was performed using the Prime PCR array (Bio-Rad) according to manufacturer’s protocol. AR primer used is forward primer; 5-GGTGAGCAGAGTGCCCTATC-3 and reverser primer; 5-TCGGGTATTTCGCATGTCCC-3. In brief, the denaturation step was carried out at 95C for 10 seconds; the annealing step was carried out at 58C for 30 seconds, and extension step at 72C for 1 minute using the applied Biosystems 7900HT fast real-time PCR system (Applied Biosystems). Sequence Detection Systems Software v2.3 was used to identify cycle threshold (Ct) values and generate gene expression curves. All data were normalized to either GAPDH expression.