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Detection of MicroRNA-Mediated Target mRNA Cleavage and 3′-Uridylation in Human Cells by a SLA-RT-PCR Analysis

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Epitranscriptomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1870))

Abstract

MicroRNA (miRNA) plays an important role in posttranscriptional regulation of gene expression by dominantly binding to the 3′-UTR regions of target mRNAs in the miRNA-induced silencing complex (miRISC), triggering off their sequential cleavage and 3′-uridylation, facilitating their degradation, repressing target gene expression, and leading to a reduced protein output. The miRNA-mediated target mRNA cleavage activity generates cleaved mRNA fragments with varied termini, which creates major technical challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs and the resulting mRNA fragments in transition. Here we described a sensitive stem-loop array reverse transcription polymerase chain reaction (SLA-RT-PCR) approach to detect and verify the miRNA-mediated target mRNA cleavage sites by determining precise sequences at the 3′- termini of cleaved mRNA fragments and their 3′-uridylation in human cells under physiological conditions. The SLA-RT-PCR methods have been demonstrated as a sensitive, cost-efficient, and high-throughput tool to systematically detect miRNA-targeted mRNA cleavage sites and fragments with 3′-uridylation in human cells.

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Acknowledgements

This work was supported in part by the National Institutes of Health/National Cancer Institute through Specialized Program of Research Excellence (SPORE) Grant CA-070907, R01 Grants CA176568 and a Department of Defense Grant W81XWH-09-02-0139 (L.J.) and The University of Texas MD Anderson Cancer Center’s Cancer Center Support Grant CA-016672.

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Authors and Affiliations

  1. Department of Thoracic and Cardiovascular Surgery, Unit 1489, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Jing Lin & Lin Ji

Authors
  1. Jing Lin
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  2. Lin Ji
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Corresponding author

Correspondence to Lin Ji .

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Editors and Affiliations

  1. Department of Pathology, Yale University School of Medicine, New Haven, CT, USA

    Narendra Wajapeyee

  2. Yale Cancer Center, Yale University School of Medicine, New Haven, CT, USA

    Romi Gupta

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Lin, J., Ji, L. (2019). Detection of MicroRNA-Mediated Target mRNA Cleavage and 3′-Uridylation in Human Cells by a SLA-RT-PCR Analysis. In: Wajapeyee, N., Gupta, R. (eds) Epitranscriptomics. Methods in Molecular Biology, vol 1870. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8808-2_9

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  • DOI: https://doi.org/10.1007/978-1-4939-8808-2_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8807-5

  • Online ISBN: 978-1-4939-8808-2

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