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. 2014 Sep 2;11:158. doi: 10.1186/1743-422X-11-158

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© Lagatie et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Validation of the stem-loop RT-PCR miRNA assays. (A) Genomic location of the JCPyV/BKPyV encoded miRNAs. (B) Graphical representation of a pre-miRNA as precursor of the mature 5p and 3p miRNAs generated upon cleavage by Dicer (C) Sequence comparison of the three miRNAs investigated. (D-F) The assay linearity and specificity was evaluated with dilution series of three synthetic miRNAs, miR-J1-5p, miR-J1a-5p and miR-B1-5p. Each dilution series was analyzed using the three miRNA assays. (G) The assay readings of miR-J1-5p by miR-J1-5p assay, miR-J1a-5p by miR-J1a-5p assay, and miR-B1-5p by miR-B1-5p assay at concentration levels of 2.10⁴ copies/μL extract were used as the relative standards (100%) for the analysis of assay cross-reactivity.