M13 forward and reverse primers

TMPRSS2-ERG translocation variants were amplified with Taq DNA polymerase, purified with a QIAquick PCR Purification Kit (Qiagen, Düsseldorf, Germany), and cloned into the pCR2.1-TOPO vector using a TOPO TA Invitrogen Cloning kit. The resulting plasmids were used for the transformation of Escherichia coli XL1-blue cells; positive clones were grown in LB medium overnight. The recombinant plasmids were isolated by the DM miniprep method [50 (link)] and sequenced in both directions using M13 forward and reverse primers (Invitrogen) and ABI BigDye Terminator Cycle Sequencing Kit v3.1 (Thermo Fisher, Waltham, MA, USA) with a Gene Amp 9700 PCR System (Thermo Fisher, Carlsbad, CA, USA). The sequences were detected with an ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA USA). The sequences of TMPRSS2 (NM_005656) and ERG (NM_004449) were analyzed with the BLAST software (National Center for Biotechnology Information, NIH, GOV, USA).

Mitotic metaphase chromosome spreads were prepared according to Doleželová et al. (1998 (link)). Probes for 45S rDNA and 5S rDNA were prepared by labeling Radka1 (45S rDNA) and Radka2 (5S rDNA) DNA clones (Valárik et al., 2002 (link)) with digoxigenin-11-dUTP or biotin-16-dUTP (Roche Applied Science, Penzberg, Germany) using PCR with M13 forward and reverse primers (Invitrogen). Probes for tandem repeats CL18 and CL33 were amplified using specific primers (Hřibová et al., 2010 (link)) and labeled as the rDNA probes using PCR. Hybridization mixture consisting of 50% formamide, 10% dextran sulfate in 1×SSC, and 1 μg/ml of each labeled probe was added onto slides and denatured at 80°C for 3 min. The hybridization was carried out at 37°C overnight. The sites of probe hybridization were detected using anti-digoxigenin-FITC (Roche Applied Science) and streptavidin-Cy3 (Vector Laboratories, Burlingame, USA), and the chromosomes were counterstained with diamidino-2-phenylindole. The slides were examined with Axio Imager Z.2 Zeiss microscope (Zeiss, Oberkochen, Germany) equipped with Cool Cube 1 camera (Metasystems, Altlussheim, Germany) and appropriate optical filters. The capture of fluorescence signals and layers merging were performed with ISIS software (Metasystems); the final image adjustment was done in Adobe Photoshop 12.0.

Probes for 45S rDNA and 5S rDNA were prepared by labeling Radka1 DNA clone (45S rDNA) and Radka2 DNA clone (5S rDNA) [44 (link)] with digoxigenin-11-dUTP or biotin-16-dUTP (Roche Applied Science). Both probes were labeled by PCR using M13 forward and reverse primers (Invitrogen). Hybridization mixture consisting of 50% formamide, 10% dextran sulfate in 1×SSC and 1 μg/ml of each labeled probe was added onto slides and denatured at 80°C for 3 min. The hybridization was carried out at 37°C overnight. The sites of probe hybridization were detected using anti-digoxigenin-FITC (Roche Applied Science) and streptavidin-Cy3 (Vector Laboratories, Burlingame, USA), and the chromosomes were counterstained with DAPI. The slides were examined with Olympus AX70 fluorescence microscope and the images of DAPI, FITC and Cy-3 fluorescence were acquired separately with a cooled high-resolution black and white CCD camera. The camera was interfaced to a PC running the MicroImage software (Olympus, Tokyo, Japan). At least ten complete metaphases were examined for every accession.