Comprehensive Measurement of Gene Silencing Involving Endogenous MicroRNAs in Mammalian Cells

  • Masashi Fukuoka
  • Hirohiko HohjohEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1733)


MicroRNAs (miRNAs) are functional small noncoding RNAs that work as mediators in gene silencing and that play important roles in gene regulation. A number of miRNAs have been found and their expression profiles have been examined by means of various microarray systems and real-time polymerase chain reaction (PCR) systems. Conventional microarrays as well as real-time PCR are able to detect existing miRNAs, in which inactive miRNAs that hardly contribute to gene silencing may be also contained. Here, we describe a comprehensive miRNA bioassay system with reporter genes for the detection of active miRNAs that are present in the RNA-induced silencing complexes, and actually working as mediators in gene silencing.

Key words

Active miRNAs Comprehensive bioassay Luciferase reporter gene psiCHECK2 vector Heat shock 


  1. 1.
    He L, Hannon GJ (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5:522–531CrossRefPubMedGoogle Scholar
  2. 2.
    Ambros V (2004) The functions of animal microRNAs. Nature 431:350–355CrossRefPubMedGoogle Scholar
  3. 3.
    Filipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9:102–114CrossRefPubMedGoogle Scholar
  4. 4.
    Stefani G, Slack FJ (2008) Small non-coding RNAs in animal development. Nat Rev Mol Cell Biol 9:219–230CrossRefPubMedGoogle Scholar
  5. 5.
    Gorospe M, Abdelmohsen K (2011) MicroRegulators come of age in senescence. Trends Genet 27:233–241CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Takahashi M, Eda A, Fukushima T, Hohjoh H (2012) Reduction of type IV collagen by upregulated miR-29 in normal elderly mouse and klotho-deficient, senescence-model mouse. PLoS One 7:e48974CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Lee Y, Kim M, Han J, Yeom KH, Lee S et al (2004) MicroRNA genes are transcribed by RNA polymerase II. EMBO J 23:4051–4060CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W et al (2002) Identification of tissue-specific microRNAs from mouse. Curr Biol 12:735–739CrossRefPubMedGoogle Scholar
  9. 9.
    Liu CG, Calin GA, Meloon B, Gamliel N, Sevignani C et al (2004) An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues. Proc Natl Acad Sci U S A 101:9740–9744CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Babak T, Zhang W, Morris Q, Blencowe BJ, Hughes TR (2004) Probing microRNAs with microarrays: tissue specificity and functional inference. RNA 10:1813–1819CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Hohjoh H, Fukushima T (2007) Expression profile analysis of microRNA (miRNA) in mouse central nervous system using a new miRNA detection system that examines hybridization signals at every step of washing. Gene 391:39–44CrossRefPubMedGoogle Scholar
  12. 12.
    Hohjoh H, Fukushima T (2007) Marked change in microRNA expression during neuronal differentiation of human teratocarcinoma NTera2D1 and mouse embryonal carcinoma P19 cells. Biochem Biophys Res Commun 362:360–367CrossRefPubMedGoogle Scholar
  13. 13.
    Eda A, Tamura Y, Yoshida M, Hohjoh H (2009) Systematic gene regulation involving miRNAs during neuronal differentiation of mouse P19 embryonic carcinoma cell. Biochem Biophys Res Commun 388:648–653CrossRefPubMedGoogle Scholar
  14. 14.
    Eda A, Takahashi M, Fukushima T, Hohjoh H (2011) Alteration of microRNA expression in the process of mouse brain growth. Gene 485:46–52CrossRefPubMedGoogle Scholar
  15. 15.
    Fukuoka M, Yoshida M, Eda A, Takahashi M, Hohjoh H (2014) Gene silencing mediated by endogenous microRNAs under heat stress conditions in mammalian cells. PLoS One 9:e103130CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.National Institute of Neuroscience, NCNPTokyoJapan

Personalised recommendations