Transcriptor 1st strand cdna kit

The 5′ proximal part of VP1 region was amplified and sequenced, amino acid position 67 to position 133 relative to the start of the VP1 reading frame of the Fermon strain of EV‐D68 (AY426531). Briefly, cDNA synthesis was performed with Transcriptor 1st Strand cDNA Kit (Roche Diagnostics, Mannheim Germany), according to manufacturer's instructions. Primer pairs as described by Nix et al.16, 2006 were used. The nested PCR product (375 bp) was analysed on a 1% agarose gel using a 100‐bp molecular weight marker (Roche, Mannheim, Germany) as a size reference. Amplicons were purified using the ExoSAP‐IT enzyme system (USB Corporation, Cleveland OH, USA) and sequenced using the Big Dye terminator version 3.1 cycle Sequencing Ready Reaction kit (Life Technologies, Foster City, CA, USA). Sequences were assembled using Sequencher^® version 5 (Gene Codes Corporation , Ann Arbor, MI, USA).

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′.