title: A contaminant-free assessment of Endogenous Retroviral RNA in human plasma.
abstract: Endogenous retroviruses (ERVs) comprise 6-8% of the human genome. HERVs are silenced in most normal tissues, up-regulated in stem cells and in placenta but also in cancer and HIV-1 infection. Crucially, there are conflicting reports on detecting HERV RNA in non-cellular clinical samples such as plasma that suggest the study of HERV RNA can be daunting. Indeed, we find that the use of real-time PCR in a quality assured clinical laboratory setting can be sensitive to low-level proviral contamination. We developed a mathematical model for low-level contamination that allowed us to design a laboratory protocol and standard operating procedures for robust measurement of HERV RNA. We focus on one family, HERV-K HML-2 (HK2) that has been most recently active even though they invaded our ancestral genomes almost 30 millions ago. We extensively validated our experimental design on a model cell culture system showing high sensitivity and specificity, totally eliminating the proviral contamination. We then tested 236 plasma samples from patients infected with HIV-1, HCV or HBV and found them to be negative. The study of HERV RNA for human translational studies should be performed with extensively validated protocols and standard operating procedures to control the widespread low-level human DNA contamination.
authors.affiliation: Department of Zoology, University of Oxford, Oxford, United Kingdom.
authors.forename: Timokratis
authors.initials: T
authors.lastname: Karamitros
authors.affiliation: Department of Hygiene, Epidemiology and Medical Statistics, Medical School, University of Athens, Athens, Greece.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Paraskevis
authors.affiliation: Department of Hygiene, Epidemiology and Medical Statistics, Medical School, University of Athens, Athens, Greece.
authors.forename: Angelos
authors.initials: A
authors.lastname: Hatzakis
authors.affiliation: First Department of Internal Medicine, Medical School, University of Athens, Athens, Greece.
authors.forename: Mina
authors.initials: M
authors.lastname: Psichogiou
authors.affiliation: Academic Department of Internal Medicine-Hepatogastroenterology, General and Oncology Hospital 'Agioi Anargyroi', Athens, Greece.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Elefsiniotis
authors.affiliation: Department of Zoology, University of Oxford, Oxford, United Kingdom.
authors.forename: Tara
authors.initials: T
authors.lastname: Hurst
authors.affiliation: Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Anna-Maria
authors.initials: AM
authors.lastname: Geretti
authors.affiliation: Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Apostolos
authors.initials: A
authors.lastname: Beloukas
authors.affiliation: Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: John
authors.initials: J
authors.lastname: Frater
authors.affiliation: Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: Klenerman
authors.affiliation: Department of Zoology, University of Oxford, Oxford, United Kingdom.
authors.forename: Aris
authors.initials: A
authors.lastname: Katzourakis
authors.affiliation: Department of Zoology, University of Oxford, Oxford, United Kingdom.
authors.forename: Gkikas
authors.initials: G
authors.lastname: Magiorkinis
mesh_terms: D001483:Base Sequence; D004279:DNA, Viral / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D020077:Endogenous Retroviruses / Q000235:genetics*; D004777:Environment; D005260:Female; D016678:Genome; D015658:HIV Infections / Q000821:virology; D006801:Humans; D008297:Male; D008954:Models, Biological; D015335:Molecular Probes / Q000737:chemistry; D009691:Nucleic Acid Denaturation; D011336:Probability; D012367:RNA, Viral / Q000097:blood*; D012015:Reference Standards; D012680:Sensitivity and Specificity
chemical_list: D004279:DNA, Viral; D015335:Molecular Probes; D012367:RNA, Viral; D003851:Deoxyribonucleases