Mouse DNA contamination in human tissue tested for XMRV

Abstract

Background: We used a PCR-based approach to study the prevalence of genetic sequences related to a gammaretrovirus, xenotropic murine leukemia virus-related virus, XMRV, in human prostate cancer. This virus has been identified in the US in prostate cancer patients and in those with chronic fatigue syndrome. However, with the exception of two patients in Germany, XMRV has not been identified in prostate cancer tissue in Europe. Most putative associations of new or old human retroviruses with diseases have turned out to be due to contamination. We have looked for XMRV sequences in DNA extracted from formalin-fixed paraffin- embedded prostate tissues. To control for contamination, PCR assays to detect either mouse mitochondrial DNA (mtDNA) or intracisternal A particle (IAP) long terminal repeat DNA were run on all samples, owing to their very high copy number in mouse cells.

Results: In general agreement with the US prevalence, XMRV-like sequences were found in 4.8% of prostate cancers. However, these were also positive, as were 21.5% of XMRV-negative cases, for IAP sequences, and many, but not all were positive for mtDNA sequences.

Conclusions: These results show that contamination with mouse DNA is widespread and detectable by the highly sensitive IAP assay, but not always with less sensitive assays, such as murine mtDNA PCR. This study highlights the ubiquitous presence of mouse DNA in laboratory specimens and offers a means of rigorous validation for future studies of murine retroviruses in human disease.

Figure 1

Nested PCR on DNA extracted from FFPE tissue of prostate cancer patients. Figure (a) shows samples that produced a PCR product of the expected size using primers specific for XMRV (lanes 1-5). UK patient 308 and UK 244 (Lane 1 and 2); Thailand 1 and Thailand 2 (Lane 3 and Lane 4); Korea 62 (Lane 5). Lanes 6-8 show samples in which XMRV was not detected. Thailand 3 (Lane 6); Korea 60 (Lane 7); Korea 61(Lane 8); Positive control pXMRV produces a strong band (lane 9). Promega 200 bp DNA step ladder (lane 10). Lanes 11-13 show water negative controls. Figure (b) shows β-globin control PCR used to demonstrate the presence of human DNA in each sample (lanes 1-8, as above). Expected size was 104 bp. Positive control LNCaP DNA is shown in lane 9. Lanes 11-13 show negative water controls. All patient samples tested showed a positive signal for β-globin. Figure (c) shows the IAP PCR result for the same samples (lanes 1-5 IAP positive, lanes 6-8 IAP negative). Positive control McCoy cell DNA is shown in lane 9. Invitrogen 100 bp DNA step ladder (lane 10). Lanes 11-13 show negative water controls. Figure (d) shows the mtDNA PCR results for the same sample (lane 1 mtDNA positive, lanes 2-8 mtDNA negative) Positive control McCoy cell DNA is shown in lane 9. Invitrogen 100 bp DNA step ladder (lane 10). Lanes 11-13 show negative water controls.

Figure 2

Sequence alignment of XMRV LTR from 7 prostate cancer patients. The gag leader primer set XMRV-R-I/XMRV-F-O bind either side of the XMRV specific deletion. Sequences were aligned against VP62 [Genbank:EF185282], MLV-releated virus CFS isolate CSF-type1 ([Genbank:HM630562], as described in Lo [17]) and mouse strain 129X1/SvJ [Genbank AAHY01591888.1]. The alignment was conducted with clustalW using BioEdit v7.0.5.3. Binding sites of primers XRMV-R-I and XMRV-F-O are shown. The asterisk shows the location of the A > G mutation.