Direct zol rna microprep extraction kit

Total RNA was extracted with a Direct-zol RNA MicroPrep Extraction kit (Zymo Research) according to manufacturer’s instructions. Then, cDNA was synthesized using Superscript IV, First-Strand Synthesis System kit (Invitrogen). The RT-qPCR technique was performed using TaqMan Fast Advanced Master Mix (Applied Biosystems) and TaqMan probes (20×) according to the manufacturer’s protocol. The list of genes: Assay name: POSTN, Assay ID: Hs0166750, PTHLH, Assay ID: Hs00174969, NOV (CCN3), Assay ID: Hs00159631 and housekeeping gene GAPDH, Assay ID: Hs02786624. The total reaction volume was 10 µL per well. RT-qPCR reaction was run in triplicates using Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument under default conditions (95°C for 10 min and then PCR reaction: 40 cycles of 95°C, 15 s and 60°C, 1 min).

Total RNA was extracted from 21 samples of CRTD5-derived NPCs and cortical mature and immature neurons using the Direct-zol RNA Micro Prep Extraction Kit according to the manufacturer’s instructions (2062, Zymo Research). RNA quality control was done by Bioanalyzer using RNA Nano 6000 assay and RNA quantity was quantified by Qubit 2.0 (Thermo Fisher Scientific, USA) to measure the RNA concentration. RNA purity was assessed by determination of the ratio for absorbance at 260 nm vs. absorbance at 280 nm (A260 nm/A280 nm) using a NanoDrop spectrophotometer and Agilent RNA 6000 Nano Kit (5067–1511, Agilent, CA, USA) as per the manufacturer’s instructions. The yields of RNA were measured using NanoDrop and Qubit. In addition, the integrity of the RNA was assessed using the 2100 Bioanalyzer by electrophoresis using the RIN-values obtained by gel electrophoresis.