title: Development of a Low-Cost Stem-Loop Real-Time Quantification PCR Technique for EBV miRNA Expression Analysis.
abstract: MicroRNAs (miRNAs) are short, single stranded, non-coding RNA molecules. They are produced by many different species and are key regulators of several physiological processes. miRNAs are also encoded by the genomes of multiple virus families, such as herpesvirus family. In particular, miRNAs from Epstein Barr virus were found at high concentrations in different associated pathologies, such as Burkitt's lymphoma, Hodgkin disease, and nasopharyngeal carcinoma. Thanks to their stability, these molecules could possibly serve as biomarkers for EBV-associated diseases. In this study, a stem-loop real-time PCR for miR-BART2-5p, miR-BART15, and miR-BART22 EBV miRNAs detection and quantification has been developed. Evaluation of these miRNAs in 31 serum samples (12 from patients affected by primary immunodeficiency, 9 from X-linked agammaglobulinemia and 10 from healthy subjects) has been carried out. The amplification performance showed a wide dynamic range (10(8)-10(2) copies/reaction) and sensibility equal to 10(2) copies/reaction for all the target tested. Serum samples analysis, on the other hand, showed a statistical significant higher level of miR-BART22 in primary immunodeficiency patients (P = 0.0001) compared to other groups and targets. The results confirmed the potential use of this assay as a tool for monitoring EBV-associated disease and for miRNAs expression profile analysis.
authors.affiliation: Department of Public Health and Pediatric Sciences, Medical School, University of Turin, Turin, Italy. massimiliano.bergallo@unito.it.|Laboratory of Cytoimmunodiagnostics, University Hospital City of Science and Health of Turin, Regina Margherita Children's Hospital, Turin, Italy. massimiliano.bergallo@unito.it.
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Bergallo
authors.affiliation: Department of Public Health and Pediatric Sciences, Medical School, University of Turin, Turin, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Merlino
authors.affiliation: S.S. Immunorheumatology, S.C. Pediatria 2, University Hospital City of Science and Health of Turin, Regina Margherita Children's Hospital, Turin, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Montin
authors.affiliation: Department of Public Health and Pediatric Sciences, Medical School, University of Turin, Turin, Italy.|Laboratory of Cytoimmunodiagnostics, University Hospital City of Science and Health of Turin, Regina Margherita Children's Hospital, Turin, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Galliano
authors.affiliation: Laboratory of Cytoimmunodiagnostics, University Hospital City of Science and Health of Turin, Regina Margherita Children's Hospital, Turin, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Gambarino
authors.affiliation: Pediatric Onco-Hematology, Stem Cell Transplantation and Cellular Therapy Division, City of Science and Health of Turin, Regina Margherita Children's Hospital, Turin, Italy.
authors.forename: Katia
authors.initials: K
authors.lastname: Mareschi
authors.affiliation: Pediatric Onco-Hematology, Stem Cell Transplantation and Cellular Therapy Division, City of Science and Health of Turin, Regina Margherita Children's Hospital, Turin, Italy.
authors.forename: Franca
authors.initials: F
authors.lastname: Fagioli
authors.affiliation: Department of Public Health and Pediatric Sciences, Medical School, University of Turin, Turin, Italy.|S.S. Immunorheumatology, S.C. Pediatria 2, University Hospital City of Science and Health of Turin, Regina Margherita Children's Hospital, Turin, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Montanari
authors.affiliation: Department of Public Health and Pediatric Sciences, Medical School, University of Turin, Turin, Italy.|Pediatric Onco-Hematology, Stem Cell Transplantation and Cellular Therapy Division, City of Science and Health of Turin, Regina Margherita Children's Hospital, Turin, Italy.
authors.forename: Silvana
authors.initials: S
authors.lastname: Martino
authors.affiliation: Department of Public Health and Pediatric Sciences, Medical School, University of Turin, Turin, Italy.|Laboratory of Cytoimmunodiagnostics, University Hospital City of Science and Health of Turin, Regina Margherita Children's Hospital, Turin, Italy.|S.S. Immunorheumatology, S.C. Pediatria 2, University Hospital City of Science and Health of Turin, Regina Margherita Children's Hospital, Turin, Italy.
authors.forename: Pier-Angelo
authors.initials: PA
authors.lastname: Tovo
mesh_terms: D000361:Agammaglobulinemia / Q000097:blood / Q000821:virology; D040181:Genetic Diseases, X-Linked / Q000097:blood / Q000821:virology; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007153:Immunologic Deficiency Syndromes / Q000097:blood / Q000821:virology; D055029:Inverted Repeat Sequences; D035683:MicroRNAs / Q000097:blood* / Q000737:chemistry; D012367:RNA, Viral / Q000097:blood / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D035683:MicroRNAs; D012367:RNA, Viral
keywords: EBV; EBV-associated pathologies; MGB; Stem-loop real-time PCR; miRNA