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Development of a Low-Cost Stem-Loop Real-Time Quantification PCR Technique for EBV miRNA Expression Analysis

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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 (108–102 copies/reaction) and sensibility equal to 102 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.

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Acknowledgments

The authors thank the colleagues of the Department of Public Health and Pediatrics. The study was sponsored by Fondazione Giovanni Goria.

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Authors and Affiliations

  1. Department of Public Health and Pediatric Sciences, Medical School, University of Turin, Turin, Italy

    Massimiliano Bergallo, Chiara Merlino, Ilaria Galliano, Paola Montanari, Silvana Martino & Pier-Angelo Tovo

  2. Laboratory of Cytoimmunodiagnostics, University Hospital City of Science and Health of Turin, Regina Margherita Children’s Hospital, Turin, Italy

    Massimiliano Bergallo, Ilaria Galliano, Stefano Gambarino & Pier-Angelo Tovo

  3. S.S. Immunorheumatology, S.C. Pediatria 2, University Hospital City of Science and Health of Turin, Regina Margherita Children’s Hospital, Turin, Italy

    Davide Montin, Paola Montanari & Pier-Angelo Tovo

  4. Pediatric Onco-Hematology, Stem Cell Transplantation and Cellular Therapy Division, City of Science and Health of Turin, Regina Margherita Children’s Hospital, Turin, Italy

    Katia Mareschi, Franca Fagioli & Silvana Martino

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  1. Massimiliano Bergallo
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  2. Chiara Merlino
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Correspondence to Massimiliano Bergallo.

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Bergallo, M., Merlino, C., Montin, D. et al. Development of a Low-Cost Stem-Loop Real-Time Quantification PCR Technique for EBV miRNA Expression Analysis. Mol Biotechnol 58, 540–550 (2016). https://doi.org/10.1007/s12033-016-9951-0

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  • DOI: https://doi.org/10.1007/s12033-016-9951-0

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