TCQA promoted differentiation of hNSCs at least toward the neuronal lineage and suggests the possibility of using TCQA to promote neurogenesis.
When differentiation is induced by growth factor withdrawal, decreased expression of stemness gene and increased expression of NSC fate-promoting genes can be observed. Therefore, we evaluated the effect of TCQA on global gene expression in hNSCs during differentiation at 24h to elucidate the neurogenesis-promoting effects of TCQA.
Overall design: The total RNA was extracted from the cells using ISOGEN kit (Nippon Gene Co. Ltd., Toyama, Japan) following manufacturer’s instructions. Total RNA was quantified and assessed for quality with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Double-stranded cDNA was synthesized from 100 ng of total RNA with the GeneAtlas 3’ IVT Express Kit (Affymetrix Inc., Santa Clara, CA, USA). Biotin-labeled amplified RNA (aRNA) was synthesized by in vitro transcription using the GeneChip 3’ IVT Express Kit (Affymetrix Inc., Santa Clara, CA, USA). A total of 9.4 mg of purified aRNA was fragmented using the GeneAtlas 3’ IVT Express Kit and was hybridized for 16 h at 45°C using GeneChip MG-430 PM microarray (Affymetrix Inc., Santa Clara, CA, USA). The chip was washed and stained in the Gene Atlas Fluidics Station 400 (Affymetrix Inc., Santa Clara, CA, USA) and then the resulting image was scanned using the GeneAtlas Imaging Station (Affymetrix Inc., Santa Clara, CA, USA). Data analysis was performed using the Partek Express software (Partek Inc., St. Louis, MO, USA) provided by Affymetrix as part of their GeneAtlas system. Compared with the non-treated cells, fold change in expression in the TCQA-treated group was calculated and converted to log 2 data.
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