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

  • Lara Stevanato
  • Caroline Hicks
  • Lavaniya Thanabalasundaram
  • John D. Sinden
Protocol
Part of the Neuromethods book series (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.

Keywords

Clinical grade neural stem cells In vitro differentiation miRNA profiling and effects miRNA target validation Three dimensional culture 

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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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Lara Stevanato
    • 1
  • Caroline Hicks
    • 1
  • Lavaniya Thanabalasundaram
    • 1
  • John D. Sinden
    • 1
  1. 1.ReNeuronPencoed Business ParkBridgendUK

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