Comprehensive NPC Differentiation Analysis

One million NPCs were pelleted for RNA extraction and qRTPCR analysis. For neuronal differentiation, 4x 10⁵ NPCs were seeded into one well of a 6 well plate for 4 weeks, and were then collected for RNA extraction. RNA was extracted using the RNeasy Mini kit (Qiagen) and reverse transcribed using the High Capacity RNA to cDNA kit (Thermo Fisher Scientific). For general differentiation marker analysis, qPCR was carried out using custom designed Taqman microfluidic cards (Thermo Fisher Scientific) containing 22 pluripotent, multipotent, neuronal and glial markers, as well as endogenous controls 18s ribosomal RNA and GAPDH[30 (link)]. Standard Taqman qPCR was used for the analysis of cellular stress-associated genes CDKN1a, SCD, CCNG2 and DDIT3, using ActB as endogenous control.

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Bowles K.R., T. C. W. J., Qian L., Jadow B.M, & Goate A.M. (2019). Reduced variability of neural progenitor cells and improved purity of neuronal cultures using magnetic activated cell sorting. PLoS ONE, 14(3), e0213374.

Publication 2019

RNeasy Mini kit High Capacity RNA to cDNA kit Taqman microfluidic cards

18s ribosomal rna Cdkn1a Cdna Cellular Ddit3 Differentiation marker Gapdh Genes Glial Neuronal

Corresponding Organization : Allen Institute for Brain Science

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Variable analysis

independent variables

Time in culture (4 weeks) for neuronal differentiation

dependent variables

Gene expression of pluripotent, multipotent, neuronal and glial markers
Gene expression of cellular stress-associated genes (CDKN1a, SCD, CCNG2, DDIT3)

control variables

Cell density (4 x 10^5 NPCs per well of a 6-well plate)
RNA extraction method (RNeasy Mini kit)
RNA reverse transcription method (High Capacity RNA to cDNA kit)
QPCR method (Taqman microfluidic cards and standard Taqman qPCR)
Endogenous controls (18s rRNA and GAPDH for general differentiation markers, ActB for cellular stress-associated genes)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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