Quantitative RT-PCR Analysis of Neural Genes

Total RNA was isolated from cell cultures using the TRI reagent (MRC, Inc., Cincinnati, OH), and 2 µg of RNA was reverse-transcribed into cDNA using random hexamer primers (Bioneer, Daejeon, Korea) and Superscript III reverse transcriptase (Invitrogen, Grand Island, NY) in a thermal cycler (Eppendorf, Happauge, NY) according to the manufacturer's instructions. Quantitative RT-PCR was carried out in a total volume of 10 µL containing 5 µL of LightCycler® 480 SYBR Green I Master (Roche Diagnostics Ltd., Rotkreuz, Switzerland), 0.5 µM of each primer and 2.5 µL of 1∶10 diluted cDNA using LightCycler® 480 instrument (Roche Diagnostics Ltd). The cycling conditions were: 95°C for 5 min, followed by 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. All the samples were carried out in triplicates. The expression levels of each mRNA expression were normalized to the housekeeping gene GAPDH using LightCycler® 480 Software, Version 1.5 (Roche Diagnostics Ltd). Primer sequences for FOXG1, DLX2, NKX2.1, GAD1, calbindin2 (CALB2) and somatostatin (SST) have been previously described [26] (link). Other primer sequences were retrieved from PrimerBank Database http://pga.mgh.harvard.edu/primerbank/[27] (link), with the exception of SLC32A1 which was designed using ProbeFinder software Version 2.49 from Roche Applied Science. Sequences of primer sets were listed in Table S1.