Detection and Monitoring of MicroRNA Expression in Developing Mouse Brain and Fixed Brain Cryosections

  • Davide De Pietri Tonelli
  • Yoanne M. Clovis
  • Wieland B. Huttner
Part of the Methods in Molecular Biology book series (MIMB, volume 1092)


MicroRNAs (miRNAs) are 20−25 nucleotide long, noncoding, and single-strand RNAs that have been found in almost all organisms and shown to exert essential roles by regulating the stability and translation of target mRNAs. In mammals most miRNAs show tissue specific and developmentally regulated expression. Approximately 70 % of all miRNAs are expressed in the brain and a growing number of studies have shown that miRNAs can modulate both brain development function and dysfunction. Moreover, miRNAs have been involved in a variety of human pathologies, including cancer and diabetes and are rapidly emerging as new potential drug targets. In order to further characterize miRNA functions, it is therefore crucial to develop techniques enabling their detection in tissues (both fixed and in vivo) with single-cell resolution. Here, we describe methods for the detection/monitoring of miRNA expression, that can be applied in both developing embryos and fixed samples, which we and others have applied to the investigation of both embryonal and postnatal neurogenesis in mice, but also in zebrafish, and cell cultures.

Key words

Development Neurogenesis MicroRNAs In utero electroporation In situ hybridization 


  1. 1.
    Fietz SA, Huttner WB (2011) Cortical progenitor expansion, self-renewal and neurogenesis—a polarized perspective. Curr Opin Neurobiol 21:23–35PubMedCrossRefGoogle Scholar
  2. 2.
    Pasquinelli AE (2012) MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship. Nat Rev. Genet 13(4):271–282PubMedGoogle Scholar
  3. 3.
    De Pietri Tonelli D, Pulvers JN, Haffner C, Murchison EP, Hannon GJ, Huttner WB (2008) miRNAs are essential for survival and differentiation of newborn neurons but not for expansion of neural progenitors during early neurogenesis in the mouse embryonic neocortex. Development 135:3911–3921PubMedCrossRefGoogle Scholar
  4. 4.
    Clovis YM, Enard W, Marinaro F, Huttner WB, De Pietri Tonelli D (2012) Convergent repression of Foxp2 3’UTR by miR-9 and miR-132 in embryonic mouse neocortex: implications for radial migration of neurons. Development 139(18):3332–3342PubMedCrossRefGoogle Scholar
  5. 5.
    Nigro A, Menon R, Bergamaschi A, Clovis YM, Baldi A, Ehrmann M, Comi G, De Pietri Tonelli D, Farina C, Martino G, Muzio L (2012) MiR-30e and miR-181d control radial glia cell proliferation via HtrA1 modulation. Cell Death Dis. 3:e360. doi:10.1038/cddis. 2012.98PubMedGoogle Scholar
  6. 6.
    McNeill E, Van Vactor D (2012) MicroRNAs shape the neuronal landscape. Neuron. 75(3):363–379PubMedCrossRefGoogle Scholar
  7. 7.
    Li X, Jin P (2010) Roles of small regulatory RNAs in determining neuronal identity. Nat Rev Neurosci 11:329–338PubMedCrossRefGoogle Scholar
  8. 8.
    De Pietri Tonelli D, Calegari F, Fei JF, Nomura T, Osumi N, Heisenberg CP, Huttner WB (2006) Single-cell detection of microRNAs in developing vertebrate embryos after acute administration of a dual-fluorescence reporter/sensor plasmid. Biotechniques 41:727–732PubMedCrossRefGoogle Scholar
  9. 9.
    Obernosterer G, Martinez J, Alenius M (2007) Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections. Nat Protoc 2:1508–1514PubMedCrossRefGoogle Scholar
  10. 10.
    Trinh LA, McCutchen MD, Bonner-Fraser M, Fraser SE, Bumm LA, McCauley DW (2007) Fluorescent in situ hybridization employing the conventional NBT/BCIP chromogenic stain. Biotechniques 42:756–759CrossRefGoogle Scholar
  11. 11.
    Osumi N, Inoue T (2001) Gene transfer into cultured mammalian embryos by electroporation. Methods 24:35–42PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Davide De Pietri Tonelli
    • 1
  • Yoanne M. Clovis
    • 1
  • Wieland B. Huttner
    • 2
  1. 1.Department of Neuroscience and Brain TechnologiesIstituto Italiano di TecnologiaGenoaItaly
  2. 2.Max Planck Institute of Molecular Cell Biology and GeneticsDresdenGermany

Personalised recommendations