ETV1 Translocations in Prostate Cancer Figure 3. A complex ETV1 translocation in xenograft PC135 involves chromosomes 4, 7 and 10. Paints of chromosomes 4, 7 and 10 are green and the ETV1 BAC (Figure 1b) is red. Black arrows indicate the translocated ETV1. In the lower panel the relevant marker chromosome is boxed in red. doi:10.1371/journal.pone.0016332.g003 RNA from clinical specimens was isolated using RNA-Bee (Campro Scientific, Berlin, Germany). DNA was isolated using the DNeasy DNA Extraction kit (Qiagen, Valencia, CA, USA). RNA from cell lines was isolated using the RNeasy RNA Extraction kit (Qiagen). Breakpoint mapping The position of the fusion point in tumor 270 was mapped by long-range PCR on genomic DNA using a forward primer in the EST14 intron and reverse primer upstream of ETV1 exon 1. PCR products were separated on a 1% agarose gel and sequenced in an ABI 3100 genetic analyzer (Applied Biosystems, Carlsbad, CA, USA). Q-PCR mRNA expression was analyzed by QPCR. cDNA was prepared with MMLV-RT (Invitrogen, Carlsbad, CA, USA) and oligo(dT)12 primer. QPCR was performed in Power SYBR Green PCR Master Mix on an ABI Prism 7700 Sequence Detection System (Applied Biosystems). Amplified products were quantified relative to porphobilinogen deaminase (PBGD) by the standard curve method. For primers see Table S1. RNA ligase-mediated rapid amplification of cDNA ends 59-RLM-RACE was performed using the GeneRacer kit of Invitrogen. cDNA was amplified using the GeneRacer 59-primer and a reverse gene-specific primer (ETV1 exon 6). PCR products were analyzed on a 1.5% agarose gel, and bands were excised, purified, and sequenced. Fluorescence in situ hybridization FISH was done on 5-mm frozen tissue sections according to standard protocols with minor modifications [14]. BAC clones RP11-124L22 (ETV1), RP11-460G19 (MIPOL1), RP11-1149J13 (DGKB) were purchased from BacPac Resources (bacpac.chori.org). BACs were either digoxigenin-11-dUTP or biotin-16-dUTP (Roche, Basel, Schweiz) labeled and visualized with anti-digoxigenin FITC (Roche) or streptavidin-Alexa 594 (Invitrogen). Tissue sections were counterstained with DAPI. Images were collected on an epifluorescence microscope (Leica DM, Wetzlar, Germany) equipped with a charge-coupled device cooled camera (Photo- metrics, Tuscon, AZ, USA). For preparation of metaphase spreads, xenograft PC135 was propagated on male nude mice. Single cells were collected by mincing and filtration. Metaphase preparation and hybridization PLoS ONE | www.plosone.org 5 January 2011 | Volume 6 | Issue 1 | e16332