Protocol

Part of the series Neuromethods pp 1-15

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

  • Lara StevanatoAffiliated withReNeuron, Pencoed Business Park Email author 
  • , Caroline HicksAffiliated withReNeuron, Pencoed Business Park
  • , Lavaniya ThanabalasundaramAffiliated withReNeuron, Pencoed Business Park
  • , John D. SindenAffiliated withReNeuron, Pencoed Business Park

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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