Quantitative RT-PCR Analysis of Prostate Cell Lines

RNA from prostate cell lines (source: ATCC, mycoplasma free) was extracted using the Qiagen RNeasy mini kit (#74104), and all other RNA samples were isolated using the Qiagen AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Manchester, UK #80004), according to the manufacturer’s instructions. RNA concentration and quality was confirmed using a NanoDrop 2000 (ThermoFisher, Loughborough, UK), and cDNA was synthesised using the Transcriptor 1st strand cDNA kit (Roche, Welwyn Garden City, UK, #04379012001) following the manufacturer’s instructions. QRT-PCR reactions were performed using 10 ng of cDNA and 2x qPCRBIO SyGreen Blue Mix (PCRBiosystems, London, UK, #PB20.16) following the manufacturer’s instructions in a QuantStudio 7 QRT-PCR machine with the following primers: AR (Exon 5-6): F 5′-CCTGGCTTCCGCAACTTACAC-3′, R 5′-GGACTTGTGCATGCGGTACTCA-3′, AR-v7: F 5′-CGTCTTCGGAAATGTTATGAAGC-3′, R 5′-GAATGAGGCAA-GTCAGCCTTTCT-3′ [41 (link)], GAPDH: F 5′-ACAGTTGCCATGTAGACC-3′, R 5′-TTGAGCACAGGGTACTTTA-3′) [42 (link)], AXIN2: F 5′-TCAAGACGGTGCTTACCTGT-3′, R 5′-TGCTGCTTCTTGATGCCATCA-3′ and ASCL2: F 5′-AAAGAACCCTTGACCTGGGG-3′, R 5′-AGATCTTGGCCAGCATGGA-3′.