Development of real-time PCRs for detection and quantitation of human MMTV-like (HML) sequences HML expression in human tissues.
The human genome contains around 1000 betaretrovirus-like copies, human mouse mammary tumour virus (MMTV)-like (HML) groups 1-10, also referred to as human endogenous retrovirus "HERV-K". Despite many efforts, it is not established whether betaretroviruses, exo- or endogenous, are involved in the etiology of breast cancer, or other cancer diseases, in humans. Quantitative real-time PCR (QPCR) TaqMan-based assays for HML groups 1-7, targeting the conserved reverse transcriptase (RT) and integrase (IN) domains of the pol gene were designed. Plasmids containing the entire pol gene of HML1-7 were used as standards. The RT and IN based QPCRs could detect 10(0)-10(3) copies per PCR reaction of the plasmids. However, not all plasmids gave a signal in both RT and IN QPCRs, probably due to mismatches. Furthermore, RT and IN based HML6 specific QPCRs were developed. They were specific for amplification of transcripts for the whole HML6 group. The methods allow the monitoring in body fluids and tissues of expression of a wide range of betaretrovirus-like sequences. Betaretrovirus-like RNA was studied in normal human tissues and of HML6 in brains of multiple sclerosis (MS) patients. Brain, adrenal gland and testis had a high betaretrovirus-like expression. Multiple sclerosis plaques contained the same HML6 RNA concentration as control tissue. These assays are expected to enhance studies on involvement of betaretroviruses in physiology and disease.
Muradrasoli
Shaman
S
Section of Virology, Department of Medical Sciences, Uppsala University, Academic Hospital, 751 85 Uppsala, Sweden. shaman.muradrasoli@medsci.uu.se
Forsman
Anna
A
Hu
Lijuan
L
Blikstad
Vidar
V
Blomberg
Jonas
J
D000311:Adrenal Glands / Q000821:virology; D001483:Base Sequence; D001826:Body Fluids / Q000821:virology; D001921:Brain / Q000821:virology; D020077:Endogenous Retroviruses / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D015751:Genes, pol; D006801:Humans; D019426:Integrases / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013737:Testis / Q000821:virology; D014764:Viral Proteins / Q000235:genetics
D012367:RNA, Viral; D014764:Viral Proteins; D019426:Integrases; D012194:RNA-Directed DNA Polymerase