Analysis of posttranscriptional regulation of CCN genes

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Cells generally control the concentration of mRNA by transcriptional and posttranscriptional regulation, so the separate contributions of synthesis and degradation (“decay”) cannot be discriminated by the quantification of mRNA. To elucidate the contribution of posttranscriptional regulation, all experimental procedures for the analysis of the total transcript level, transcriptional induction, and degradation of the target mRNA are performed either individually, or in combination. From our experience, measurement of the steady-state levels of the mRNA using quantitative real-time polymerase chain reaction is an essential first step in quantifying ccn2 gene expression level. Subsequently, the effect of transcription rates should be assessed by reporter assays of the ccn2 promoter and nuclear run-on assays. Finally, the stability of ccn2 mRNAs is evaluated in the presence of a metabolic inhibitor actinomycin D, followed by mRNA degradation assays in vitro. Here, we describe the strategic methods used in a series of analyses to elucidate the possible involvement of the posttranscriptional regulatory mechanism of the ccn2 gene and show how this approach can in theory be applied to elucidating the posttranscriptional regulation of other genes belonging to the CCN family.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages187-209
Number of pages23
Volume1489
DOIs
Publication statusPublished - 2017

Publication series

NameMethods in Molecular Biology
Volume1489
ISSN (Print)10643745

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Keywords

  • 3′-Untranslated region (3′-UTR)
  • CCN
  • Gene expression
  • mRNA degradation
  • Posttranscriptional regulation

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Kondo, S., Kubota, S., & Takigawa, M. (2017). Analysis of posttranscriptional regulation of CCN genes. In Methods in Molecular Biology (Vol. 1489, pp. 187-209). (Methods in Molecular Biology; Vol. 1489). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-6430-7_19