4.10. Real-Time PCR

For RNA-Seq validation with real-time PCR, eight samples for condition were analyzed (n = 8). Specifically, four experiments were performed each including two samples of NPCs grown in standard floating conditions for 7 days, two samples of NPCs grown inside the Nichoid for 7 days, and two samples of NPCs grown inside the Nichoid and replated in standard floating condition for 7 more days. Total RNA (500 ng) was reverse transcribed using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Using gene sequences available from NCBI for target genes (http://www.ncbi.nlm.nih.gov/nucleotide, 29 May 2020) PCR oligonucleotide primers for target genes were selected and primers are reported in Table S12. This was done with the NCBI’s Primer-BLAST tool. Real-time PCR was performed with StepOnePlus^TM Real-Time PCR System (Thermo Fisher, Waltham, MA, USA) using SSOSYBR Green Supermix (Bio-Rad, Hercules, CA, USA). Genes were quantified in triplicates, GAPDH was used as housekeeping gene for mouse samples. Gene expression was calculated using the 2^−ΔΔCt method.