revolves around testing for contamination of samples from mouse DNA,
such as Section 3.5 in this study, 'Ultrasensitive assays for mouse
nucleic acid contamination'. However if the samples were potentially
contaminated with XMRV itself and not from mouse cells then there
would be no reason to believe that tests for mouse DNA would rule out
sample contamination since XMRV does not exist in mice to begin with
but rather comes from infected cell lines. These infected cell lines
contain neither IAP nor mouse mitochondrial DNA, which are two tests
currently run to check for contamination.
---------------------------------------------------------------------------=
------
http://etd.ohiolink.edu/send-pdf.cgi/Barton%20Maria.pdf?kent1309619878
EVALUATING XMRV AS AN INDICATOR OF PROSTATE CANCER RISK
A thesis submitted to Kent State University in collaboration with the
Lerner Research Institute, Cleveland Clinic in partial fulfillment of
the requirements for the degree of MASTER OF SCIENCE by Maria Barton
August, 2011
Thesis written by Maria Barton B.S., School of Chemistry UdelaR, 2007
M.S., Kent State University, 2011
Table of Contents
List of figures =85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=
=85=85=85=85......... vi
List of tables =85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=
=85=85=85=85=85.. vii
Acknowledgments=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=
=85=85.=85 viii
Hypothesis =85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=
=85=85=85=85=85.. xi
CHAPTER 1: INTRODUCTION
1.1 - XMRV history and link with prostate cancer =85=85=85=85=85=85=85=85=
=85=85..=85. 1
1.2 =96 The role of RNaseL in prostate cancer =85=85=85=85=85=85=85=85=85=
=85=85=85=85.... 3
1.3 - Identification of a novel gammaretrovirus in prostate cancer tissues =
.5
1.4 - XMRV replication-competent genome and its integration sites =85.=85. =
10
1.5 - Scope of this project =85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=
=85=85=85=85=85=85... 13
CHAPTER 2: MATERIALS AND METHODS
2.1 - Urine sample collection=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=
=85=85=85=85=85... 15
2.2 - Isolation of total RNA in patient urine specimen =85=85=85=85=85=85..=
.=85=85.. 16
2.3 - Synthesis of XMRV envelope RNA by in vitro transcription and its
use as standard in real time RT - PCR assays =85...=85=85=85=85=85=85=85=85=
.. 17
2.4 - Polymerase chain reaction
2.4.1 - Quantitative RT- PCR =85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=
=85=85=85 19
2.4.2 - Nested RT- PCR
.........................................................................
21
2.5 - Mouse mitochondrial RNA/DNA contamination assays =85=85=85=85=85=85. =
24
2.5.1 - Mitochondrial (mt) COX-2 assay =85=85=85=85=85=85=85=85=85=85=85=85=
=85 25
2.5.2 - Intracisternal A-particles (IAP) retrotransposon assay =85...... 26
CHAPTER 3: RESULTS
3.1 - Generation of standard curves by qRT - PCR and nRT - PCR using
known copy numbers of RNA=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=
=85=85 27
3.2 - Analysis of RNA samples from prostate cancer urine specimens
for the presence of XMRV by qRT - PCR =85=85=85=85=85=85=85=85=85=85=85=85.=
. 30
3.3 - Prostate cancer urine samples tested by nRT - PCR =85=85=85=85=85=85=
=85.. 34
3.4 - Partial sequences of PCR products aligned to
XMRV reference strain VP62 =85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=
=85=85.. 36
3.5 - Ultrasensitive assays for mouse nucleic acid contamination =85=85=85.=
. 38
3.6 - Data summary .=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=
=85=85=85=85=85=85... 41
CHAPTER 4: DISCUSSION
4.1 - Retrovirus associated with prostate cancer =85=85=85.=85=85=85=85=85=
=85........ 42
4.2 - XMRV and prostate carcinogenesis =85=85=85=85=85=85=85=85=85=85=85=85=
=85=85=85. 44
4.3 - Contamination due to mouse sources ruled out =85=85=85=85=85=85=85=85=
=85.. 46
4.4 - XMRV and detection in urine =85=85=85=85=85=85=85=85=85=85=85=85=85=
=85=85=85=85=85. 47
Summary =85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=
=85=85=85=85=85. 48
References=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=85=
=85=85=85=85=85... 49
Appendix
MutVar XMRV env RNA construction ..=85=85=85=85=85=85=85=85=85=85=85=85=85=
=85=85.. 57
Hypothesis
The presence of XMRV in tumor-bearing prostatic tissues and its
association with a reduced activity variant of RNASEL/HPC1 (Urisman et
al. 2006) suggested that the role of RNaseL in prostate cancer
pathogenesis is to suppress XMRV infections. Thus, we hypothesized
that detection of XMRV infections would predict prostate cancer
occurrence and possibly disease aggressiveness and might eventually
lead to novel therapeutic options (such as antivirals) or preventive
strategies (vaccines), like with other viruses such as HPV (Bharti et
al. 2009). However, current methods of monitoring XMRV infections are
cumbersome, requiring tumor tissue or biopsy material, and are not
suitable for widespread screening of populations. Prostate cancer
patient urine samples have already been used as a source of RNA for
the detection of genes of interest (Laxman et al. 2006). Our objective
was to develop a simple, ultrasensitive and non-invasive clinical
assay for the monitoring of XMRV in urine that would be highly
accurate, leading to eventual high throughput screening of patients.
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