The UK prostate specimens in this study came from two sources. Cancer tissue came from men locally referred to St Mary's Hospital in West London with symptoms of voiding dysfunction and prostate specific antigen abnormality and requiring biopsy after appropriate counselling. In addition, some patients had participated in a voluntary screening study and these provided samples that were a mixture of benign and cancer pathologies. All biopsy tissue had been stored in formalin fixed, paraffin-embedded (FFPE) blocks over a period of 3-6 years. Slices were taken from the blocks and DNA extracted as described (see Additional File 1 ; Supplementary Methods). The FFPE samples from Thailand and Korea were excess tissue from histological samples taken from new cases of both benign prostate hyperplasia and prostate cancer. These were FFPE embedded and sent to London for analysis.
We investigated the prevalence of XMRV in the UK and in the Far East, aware that the close relationship (about 94% at the nucleotide level) to other murine exogenous and endogenous retroviruses posed a problem in distinguishing XMRV from contaminating mouse DNA sequences. We were further aware that in any retrovirology laboratory MLV sequence contamination is something of an occupational hazard [19 (link)]. For these reasons, we extracted the DNA from FFPE prostate cancers, along with benign hyperplasia tissue, and PBS without tissue. We used several sets of primers [12 (link)] to test for XMRV-specific sequences, derived from the XMRV gag leader [1 (link)] which encompasses the 24 bp deletion originally thought to distinguish XMRV as a new human virus. To control for low level contamination, we included multiple no-template controls (no less than 6 in every run) and included assays with primers that would amplify murine mitochondrial DNA (mtDNA) and intracisternal A particle (IAP) LTRs. IAPs are retrotransposons present at the level of about 1000 copies of varying length per mouse genome [20 (link)].
We investigated the prevalence of XMRV in the UK and in the Far East, aware that the close relationship (about 94% at the nucleotide level) to other murine exogenous and endogenous retroviruses posed a problem in distinguishing XMRV from contaminating mouse DNA sequences. We were further aware that in any retrovirology laboratory MLV sequence contamination is something of an occupational hazard [19 (link)]. For these reasons, we extracted the DNA from FFPE prostate cancers, along with benign hyperplasia tissue, and PBS without tissue. We used several sets of primers [12 (link)] to test for XMRV-specific sequences, derived from the XMRV gag leader [1 (link)] which encompasses the 24 bp deletion originally thought to distinguish XMRV as a new human virus. To control for low level contamination, we included multiple no-template controls (no less than 6 in every run) and included assays with primers that would amplify murine mitochondrial DNA (mtDNA) and intracisternal A particle (IAP) LTRs. IAPs are retrotransposons present at the level of about 1000 copies of varying length per mouse genome [20 (link)].
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