Detection of microRNA-mediated target mRNA cleavage and 3′-uridylation in human cells by a SLA-RT-PCR analysis

Jing Lin, Lin Ji

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

LanguageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages125-136
Number of pages12
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Molecular Biology
Volume1870
ISSN (Print)1064-3745

Fingerprint

MicroRNAs
Reverse Transcription
Stem Cells
Polymerase Chain Reaction
Messenger RNA
Gene Expression Regulation
3' Untranslated Regions
Gene Expression
Costs and Cost Analysis
Proteins

Keywords

  • 3′-Uridylation
  • miRNA
  • mRNA cleavage
  • Stem-loop array reverse transcription polymerase chain reaction (SLA-RT-PCR)

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

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 Methods in Molecular Biology (pp. 125-136). (Methods in Molecular Biology; Vol. 1870). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8808-2_9

Detection of microRNA-mediated target mRNA cleavage and 3′-uridylation in human cells by a SLA-RT-PCR analysis. / Lin, Jing; Ji, Lin.

Methods in Molecular Biology. Humana Press Inc., 2019. p. 125-136 (Methods in Molecular Biology; Vol. 1870).

Research output: Chapter in Book/Report/Conference proceedingChapter

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 Methods in Molecular Biology. Methods in Molecular Biology, vol. 1870, Humana Press Inc., pp. 125-136. https://doi.org/10.1007/978-1-4939-8808-2_9
Lin J, Ji L. Detection of microRNA-mediated target mRNA cleavage and 3′-uridylation in human cells by a SLA-RT-PCR analysis. In Methods in Molecular Biology. Humana Press Inc. 2019. p. 125-136. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-8808-2_9
Lin, Jing ; Ji, Lin. / Detection of microRNA-mediated target mRNA cleavage and 3′-uridylation in human cells by a SLA-RT-PCR analysis. Methods in Molecular Biology. Humana Press Inc., 2019. pp. 125-136 (Methods in Molecular Biology).
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