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MicroRNA Technologies pp 43–57Cite as

MicroRNA Expression Profiling by PCR Array in 2D and 3D Differentiated Neural Culture Systems and Target Validation

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Part of the book series: Neuromethods ((NM,volume 128))

Abstract

MicroRNAs (miRNAs) have been proven to regulate gene expression at post-transcriptional level and are emerging as strong mediators in neural fate determination (Ambros, Nature 431(7006):350–355, 2004). Here, we evaluated appropriate 3 three dimensional (3D) substrates to differentiate human neural stem cells (hNSCs). We identified and quantified hNSC miRNA contents by PCR array. By using computational algorithms we predicted miRNA target mRNA which correlates with hNSC differentiation and performed target validation by transfection of 3 prime untranslated regions (3′UTR) dual reporter plasmids and dual luciferase assay. Despite the inherent differences between cultures, we were able to consistently show that 3D topography promotes differentiation of hNSCs through modulation of miRNAs associated with cell proliferation and maintenance of stemness.

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Abbreviations

1 W:

1 Week

2D:

Two dimensional

3 W:

3 Weeks

3′UTR:

3 Prime untranslated region

3D:

Three dimensional

4-OHT:

4-Hydroxytamoxifen

bFGF:

Basic fibroblast growth factor

DCX:

Doublecortin

EGF:

Epidermal growth factor receptor

GALC:

Galactosylceramidase

GFAP:

Glial fibrillary acidic protein

HNSC:

Human neural stem cell

Hsa-miR:

Human miRNA

ICC:

Immunocytochemistry

MAP2:

Microtubule-associated protein 2

miRNA:

MicroRNA

mRNA:

Messenger RNA

QRT-PCR:

Real-time reverse transcription PCR

S100B:

S100 calcium binding protein B

TUBB3:

Tubulin, beta 3 class III

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Acknowledgement

This study was supported by ReNeuron (RENE.L). We acknowledge Julie Heward for helping in the preparation of hNSCs.

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Authors and Affiliations

  1. ReNeuron, Pencoed Business Park, Pencoed, Bridgend, CF35 5HY, UK

    Lara Stevanato, Caroline Hicks, Lavaniya Thanabalasundaram & John D. Sinden

Authors
  1. Lara Stevanato
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  2. Caroline Hicks
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  3. Lavaniya Thanabalasundaram
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  4. John D. Sinden
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Corresponding author

Correspondence to Lara Stevanato .

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Editors and Affiliations

  1. Institute of Human Genetics, University of Cologne, Cologne, Germany

    Min Jeong Kye

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Stevanato, L., Hicks, C., Thanabalasundaram, L., Sinden, J.D. (2017). MicroRNA Expression Profiling by PCR Array in 2D and 3D Differentiated Neural Culture Systems and Target Validation. In: Kye, M. (eds) MicroRNA Technologies. Neuromethods, vol 128. Humana Press, New York, NY. https://doi.org/10.1007/7657_2017_1

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  • DOI: https://doi.org/10.1007/7657_2017_1

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7173-2

  • Online ISBN: 978-1-4939-7175-6

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