Quantifying Neural Lineage Markers

At different time points of differentiation, RNA samples were extracted by using High Pure RNA Isolation Kit (Roche) and converted into complementary cDNA with Transcriptor High Fidelity cDNA Synthesis Kit (Roche). Real-time quantitative PCR (qPCR) was performed using the StepOne^TM or the ViiA^TM 7 RT-PCR Systems (Applied BioSystems). Taqman^® Gene Expression Assays (20X, Applied Biosystems) were selected for NANOG, PAX6, TBR1, DLX2, NKX2.1, LHX6, FOXG1 and GAPDH. DLL1, HES5, PARVALBUMIN (PV), VGLUT1 and GAPDH analysis were performed using the SYBR Green Master Mix (Nzytech). The results were analyzed with the StepOne^TM or the QuantStudio^TM RT-PCR Software. All PCR reactions were done in duplicate or triplicate and then normalized to the housekeeping gene GAPDH. The fold change was calculated using the 2^ΔCt method and in some graphical results it was calculated relatively to the control condition levels obtained. The representative heatmaps were generated using the web tool Clustvis (Metsalu and Vilo, 2015 (link)).