RT-PCR for AR-FL and AR-V7

Quantitative RT-PCR analysis for full length AR and AR-V7 in metastatic CRPC specimens was carried as described previously. For each tested sample, 5–10 cryosections of 6 μm thickness were prepared from frozen metastatic CRPC tissue blocks. Total RNA was extracted and purified by TRIZOL (Invitrogen, Carlsbad, CA) followed by RNeasy Mini Kit (Qiagen, Valencia, CA). Briefly, 1mL TRIZOL was added into the tube containing cryosections. Following repeated vortexing, 200 μl chloroform was added to the dissolved tissue sections. The mixture was vortexed and centrifuged at 13,000 rpm x 10 min at 4°C. The tope aqueous layer was carefully transferred to a new tube and mixed with an equal volume of 70% ethanol. The mixed lysate was onto RNeasy Mini Spin Column to further purification by RNeasy Mini Kit according to manufacturer’s instructions. The quality of total RNA was ascertained by RNA 6000 Nano kit on Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA). The RNA concentration was determined by checking UV absorbance at 260 nm on spectrophotometer (Nanodorp, Wilmington, DE). Reverse transcription (RT) (500 ng input total RNA) was performed using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. The ensuing quantitative PCR (qPCR) was performed using 1x iQ SYBR Green Supermix (Bio-Rad, Hercules, CA), 400 nM forward primer and 400 nM reverse primers on CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Primer sequences are RPL13A (control gene), full length AR (AR-FL), and AR-V7 are as follows:

RPL13A-forward: 5′-CCTGGAGGAGAAGAGGAAAGAGA-3′

RPL13A-reverse: 5′-TTGAGGACCTCTGTGTATTTGTCAA-3′

AR-FL-forward: 5′-CAGCCTATTGCGAGAGAGCTG-3′

AR-FL-reverse: 5′-GAAAGGATCTTGGGCACTTGC-3′

AR-V7-forward: 5′-CCATCTTGTCGTCTTCGGAAATGTTA-3′

AR-V7-reverse: 5′-TTTGAATGAGGCAAGTCAGCCTTTCT-3′