FISH analysis did not detect XMRV nucleic acid or closely related sequences in CWR22 primary prostate tissues.

<p>Each slide was hybridized with a probe mix consisting of XMRV-SO viral probe derived from a full-length XMRV VP62 and CEP8-SA internal control probe from the centromeric sequence of human chromosome 8. (A) representative image showing XMRV-SO orange staining in a mixture of uninfected DU145 prostate cancer cells and XMRV-infected 22Rv1; (B) the same image showing CEP8-SA aqua staining in DU145 (three copies/cell) and 22Rv1 (two copies/cell); (C – G) representative images showing XMRV-SO FISH results on tissue sections from blocks A, B, C, E, and K, respectively, from CWR22.</p

SNP Genotyping of CWR22 primary tumor, CWR22 xenograft, and 22Rv1 cells.

<p>SNP Genotyping of CWR22 primary tumor, CWR22 xenograft, and 22Rv1 cells.</p

An absence of detectable levels of XMRV DNA or closely related DNA in CWR22 primary prostate tissues was determined by real-time PCR analysis.

<p>Amplification plots from the real-time PCR analysis of (A) XMRV (FAM) signal in DNA extracted from different sections of CWR22 prostate tissues and run controls with the <i>pol</i> and <i>env</i> primer/probe sets; (B) <i>β-</i>globin (Cy5) signal during the same run.</p

An absence of detectable levels of XMRV DNA or closely related DNA in CWR22 primary prostate tissues was determined by qPCR analysis.

<p>Amplification plot of real-time qPCR analysis for the (A) detection of XMRV specific regions (<i>gag</i>, <i>pol</i> and <i>env</i>) using XMRV VP62 plasmid DNA (3,750 copies) and (B) in DNA extracted from different sections of CWR22 prostate tissues (tissue blocks A, B, C & E, each assayed in duplicate). For block C only, 1 of 2 assay for <i>env</i> was weakly positive, all other assays for <i>gag</i>, <i>pol</i> and <i>env</i> were negative. <i>RNase P</i> probes were used to detect the presence of genomic DNA in tumor tissues.</p

Summary of XMRV and Control Assays in CWR22 Prostate Samples.

*<p>Reduced input DNA amount to 17 ng, **Ct value of 45, *** Ct value of 41.</p

Detection of XMRV in Archival RNA Extracts by Microarray and PCR.

<p>NT, not tested due to lack of sample availability.</p>*<p>Urisman, <i>et al.,</i> (2006) <i>PloS Pathogens</i>, 2(3):e25.</p

Detection of XMRV in Prostate Cancer Tissues and Archival RNA Extracts by Microarray.

<p>Samples were analyzed using the ViroChip, a pan-viral DNA detection microarray (x-axis). The heat map shows a selected cluster consisting of 96 gammaretrovirus probes (y-axis) and corresponding to the same cluster observed in the 2006 study by Urisman, <i>et al </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044954#pone.0044954-Urisman1" target="_blank">[1]</a>. The red color saturation indicates the normalized magnitude of hybridization intensity. Microarrays corresponding to key samples are highlighted (arrows). Only prostate cancer samples VP35 and VP42 were found to be consistently positive for XMRV from both total and polyA RNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044954#pone.0044954-Urisman1" target="_blank">[1]</a>.</p

Proposed Model for Laboratory Contamination by XMRV.

<p>Early contamination of VP35/VP42 prostate cancer tissues and/or extracted RNA by XMRV-infected LNCaP cells resulted in mistaken identification of XMRV in association with prostate cancer.</p

Detection of XMRV in Prostate Cancer Tissues by PCR.

*<p>includes VP62(2012).</p>Δ<p>one prospectively collected sample not tested due to lack of sample availability.</p

Lack of Diversity Among XMRV Strains Detected in Laboratory Cell Lines and Prostate Cancer Tissues.

<p>By SNP analysis, single nucleotide differences between the sequences of 22Rv1-associated XMRV and XMRV genomes detected in prostate cancer tissues [VP35, VP42, and VP62(2006)] (red lollipops) are corrected by the deep sequencing coverage data (black lollipops). The depth of read coverage achieved at the nucleotide position corresponding to each SNP is displayed below the x-axis. All reads covering a given position yielded the same (corrected) nucleotide, indicating that previous nucleotide differences in published genomes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044954#pone.0044954-Urisman1" target="_blank">[1]</a> (red lollipops) are due to sequencing error. A natural A→G polymorphism in the XMRV genome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044954#pone.0044954-Sakuma1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044954#pone.0044954-Paprotka1" target="_blank">[38]</a> is present at position 790 (cyan lollipop). Note that XMRV consensus genomes associated with 22Rv1, LNCaP, and the 3 XMRV-positive prostate cancer tissues are identical.</p